Subcutaneous anti-HER2 Antibody Formulations and Uses Thereof

ABSTRACT

The present invention relates to a highly concentrated, stable pharmaceutical formulation of a pharmaceutically active anti-HER2 antibody, such as e.g. Trastuzumab (HERCEPTIN™), Pertuzumab or T-DM1, or a mixture of such antibody molecules for subcutaneous injection. In particular, the present invention relates to formulations comprising, in addition to a suitable amount of the anti-HER2 antibody, an effective amount of at least one hyaluronidase enzyme as a combined formulation or for use in form of a co-formulation. The formulations comprise additionally at least one buffering agent, such as e.g. a histidine buffer, a stabilizer or a mixture of two or more stabilizers (e.g. a saccharide, such as e.g. α,α-trehalose dihydrate or sucrose, and optionally methionine as a second stabilizer), a nonionic surfactant and an effective amount of at least one hyaluronidase enzyme. Methods for preparing such formulations and their uses thereof are also provided.

RELATED APPLICATIONS

This application is a non-provisional application claiming priority toEuropean Application No. 09167025.7 filed Jul. 31, 2009, the contents ofwhich are hereby incorporated by reference.

BACKGROUND OF THE INVENTION

The present invention relates to highly concentrated, stablepharmaceutical formulations of a pharmaceutically active anti-HER2antibody or a mixture of such antibody molecules for subcutaneousinjection. Such formulations comprise, in addition to the high amountsof anti-HER2 antibody or mixture thereof, a buffering agent, astabilizer or a mixture of two ore more stabilizing agents, a nonionicsurfactant and an effective amount of at least one hyaluronidase enzyme.The invention also relates to a process for the preparation of the saidformulation and to the uses of such formulation.

The pharmaceutical use of antibodies has increased over the past years.In many instances such antibodies are injected via the intravenous (IV)route. Unfortunately the amount of antibody that can be injected via theintravenous route is limited by the physico-chemical properties of theantibody, in particularly by its solubility and stability in a suitableliquid formulation and by the volume of the infusion fluid. Alternativeadministration pathways are subcutaneous or intramuscular injection.These injection pathways require high protein concentration in the finalsolution to be injected [Shire, S. J., Shahrokh, Z. et al., “Challengesin the development of high protein concentration formulations”, J.Pharm. Sci. 2004; 93(6): 1390-1402; Roskos, L. K., Davis C. G. et al.,“The clinical pharmacology of therapeutic antibodies”, Drug DevelopmentResearch 2004; 61(3): 108-120]. In order to increase the volume, andthereby the therapeutic dose, which can be safely and comfortablyadministered subcutaneously it has been proposed to useglycosaminoglycanase enzyme(s) in order to increase the interstitialspace into which the antibody formulation can be injected[WO2006/091871].

Examples of stable formulations of pharmaceutically active antibodiesfor therapeutic use currently on the market are as follows:

HERCEPTIN™ (Trastuzumab) is a monoclonal antibody directed against theHER2 receptor (anti HER2 antibody) which is currently marketed in Europein form of a 150 mg lyophilized powder (containing the antibody,α,α-trehalose dihydrate, L-histidine and L-histidine hydrochloride andpolysorbate 20) which is reconstituted for infusions with water forinjection to yield an injection dose of approximately 21 mg/ml. In theUSA and many other countries a multiple dosage vial containing 440 mgTrastuzumab is marketed.

AVASTIN™ (Bevacizumab) is a monoclonal antibody directed against thevascular endothelial growth factor (VEGF) which is currently marketed inEurope as a liquid formulation in two types of vials: a) 100 mgBevacizumab in 4 ml and b) 400 mg Bevacizumab in 16 ml, respectively,providing a final concentration of 25 mg/ml in water for injectioncontaining the following excipients: trehalose dihydrate, sodiumphosphate and polysorbate 20.

While the above antibody formulations have been found suitable forintravenous administration there is a desire to provide highlyconcentrated, stable pharmaceutical formulations of therapeuticallyactive antibodies for subcutaneous injection. The advantage ofsubcutaneous injections is that it allows the medical practitioner toperform it in a rather short intervention with the patient. Moreover thepatient can be trained to perform the subcutaneous injection by himself.Such self-administration is particularly useful during maintenancedosing because no hospital care is needed (reduced medical resourceutilization). Usually injections via the subcutaneous route are limitedto approximately 2 ml. For patients requiring multiple doses, severalunit dose formulations can be injected at multiple sites of the bodysurface.

The following two antibody products for subcutaneous administration arealready on the market.

HUMIRA™ (Adalimumab) is a monoclonal antibody directed against tumornecrosis factor alpha (TNF alpha) which is currently marketed in Europein form of a 40 mg dose in 0.8 ml injection volume for subcutaneousapplication (concentration: 50 mg antibody/ml injection volume).

XOLAIR™ (Omalizumab) a monoclonal antibody directed againstimmunoglobulin E (anti IgE antibody) which is currently marketed in formof a 150 mg lyophilized powder (containing the antibody, sucrose,histidine and histidine hydrochloride monohydrate and polysorbate 20)which is be reconstituted with water for subcutaneous injection to yielda 125 mg/ml injection dose.

No highly concentrated, stable pharmaceutical anti-HER2 antibodyformulation suitable for subcutaneous administration is currentlyavailable on the market. There is therefore a desire to provide suchhighly concentrated, stable pharmaceutical formulations oftherapeutically active antibodies for subcutaneous injection.

The injection of parenteral drugs into the hypodermis is generallylimited to volumes of less than 2 ml due to the viscoelastic resistanceto hydraulic conductance in the subcutaneous (SC) tissue, due to thegenerated backpressure upon injection [Aukland K. and Reed R.,“Interstitial-Lymphatic Mechanisms in the control of Extracellular FluidVolume”, Physiology Reviews”, 1993; 73:1-78], as well as due to theperceptions of pain.

The preparation of high concentration protein formulations is ratherchallenging and there is a need to adapt each formulation to theparticular proteins used because each protein has a differentaggregation behavior. Aggregates are suspected to cause immunogenicityof therapeutic proteins in at least some of the cases. Immunogenicreaction against protein or antibody aggregates may lead to neutralizingantibodies which may render the therapeutic protein or antibodyineffective. It appears that the immunogenicity of protein aggregates ismost problematic in connection with subcutaneous injections, wherebyrepeated administration increases the risk of an immune response.

While antibodies have a very similar overall structure, such antibodiesdiffer in the amino acid composition (in particular in the CDR regionsresponsible for the binding to the antigen) and the glycosylationpattern. Moreover there may additionally be post-translationalmodifications such as charge and glycosylation variants. In theparticular case of anti-HER2 antibodies such post-translationalmodifications have been described e.g. for the humanized monoclonalantibody humMAb4D5-8 (=Trastuzumab). Particular purification methods forthe removal of e.g. acidic variants have been developed and compositionscomprising a reduced amount of acidic variants (predominantly deamidatedvariants wherein one or more asparagine residue(s) of the originalpolypeptide have been converted to aspartate, i.e. the neutral amideside chain has been converted to a residue with an overall acidiccharacter) have first been provided by Basey, C. D and Blank, G. S. inWO99/57134.

Stable lyophilized antibody formulations comprising a lyoprotectant, abuffer and a surfactant have been described by Andya et al. (WO 97/04801and U.S. Pat. Nos. 6,267,958, 6,685,940, 6,821,151 and 7,060,268).

WO 2006/044908 provides antibody formulations, including monoclonalantibodies formulated in histidine-acetate buffer, pH 5.5 to 6.5,preferably 5.8 to 6.2.

The problem to be solved by the present invention is therefore toprovide novel highly concentrated, stable pharmaceutical formulations ofa pharmaceutically active anti-HER2 antibody or a mixture of anti-HER2antibody molecules for subcutaneous injection. Such formulationscomprise, in addition to the high amounts of anti-HER2 antibody oranti-HER2 antibody mixture, a buffering agent, a stabilizer or a mixtureof two or more stabilizers, a nonionic surfactant and an effectiveamount of at least one hyaluronidase enzyme. The preparation ofhighly-concentrated antibody formulations is challenging because of apotential increase in viscosity at higher protein concentration and apotential increase in protein aggregation, a phenomenon that is per seconcentration-dependent. High viscosities negatively impact the processability (e.g. pumping and filtration steps) of the antibody formulationsand the administration (e.g. the syringe ability). By the addition ofexcipients high viscosities could be decreased in some cases. Controland analysis of protein aggregation is an increasing challenge.Aggregation is potentially encountered during various steps of themanufacturing process, which include fermentation, purification,formulation and during storage. Different factors, such as temperature,protein concentration, agitation stress, freezing and thawing, solventand surfactant effects, and chemical modifications, might influence theaggregation behavior of a therapeutic protein. During development of ahighly concentrated antibody formulation the aggregation tendency of theprotein has to be monitored and controlled by the addition of variousexcipients and surfactants [Kiese S. et al., J. Pharm. Sci., 2008;97(10); 4347-4366]. The challenge to prepare suitable highlyconcentrated, stable pharmaceutical formulation of the pharmaceuticallyactive anti-HER2 antibody in accordance with the present invention isincreased by the fact that two different proteins have to be formulatedin one liquid formulation in such a way that the formulation remainsstable over several weeks and the pharmaceutically active ingredientsremain active during proper storage.

SUMMARY OF THE INVENTION

In a first aspect the present invention provides highly concentrated,stable pharmaceutical formulations of a pharmaceutically activeanti-HER2 antibody or a mixture of such antibody molecules forsubcutaneous injection, which are ready for use.

More particularly the highly concentrated, stable pharmaceuticalformulation of a pharmaceutically active anti-HER2 antibody formulationof the present invention comprises:

-   -   about 50 to 350 mg/ml anti-HER2 antibody;    -   about 1 to 100 mM of a buffering agent providing a pH of        5.5±2.0;    -   about 1 to 500 mM of a stabilizer or a mixture of two or more        stabilizers, whereby optionally methionine is present as a        secondary stabilizer e.g. in a concentration of 5 to 25 mM;    -   0.01 to 0.1% of a nonionic surfactant; and    -   an effective amount of at least one hyaluronidase enzyme.

In a further aspect the present invention provides for use of aformulation for the preparation of a medicament useful for treating adisease or disorder amenable to treatment with an anti-HER2 antibodysuch as e.g. cancer or a non-malignant disease in a subject comprisingadministering the formulation described herein to a subject in an amounteffective to treat the said disease or disorder. The anti-HER2 antibodycan be co-administered concomitantly or sequentially with achemotherapeutic agent.

In another aspect the present invention there are provided methods oftreating a disease or disorder which is amenable to treatment with ananti-HER2 antibody (e.g. cancer or a non-malignant disease) in a subjectcomprising administering the formulation described herein to a subjectin an amount effective to treat the said disease or disorder. The canceror a non-malignant disease will generally involve HER2-expressing cells,such that the HER2 antibody in the therapeutic pharmaceutical SCformulation in accordance with the present invention is able to bind tothe affected cells.

The present invention also provides pharmaceutical compositionsconsisting of a highly concentrated, stable pharmaceutical formulationof a pharmaceutically active anti-HER2 antibody or a mixture of suchantibody and a suitable amount of at least one hyaluronidase enzyme inthe form of a kit comprising both injection components and suitableinstructions for their subcutaneous administration.

A further aspect of the present invention relates to injection devicescomprising a highly concentrated, stable pharmaceutical formulation inaccordance with the present invention. Such formulation may consist of apharmaceutically active anti-HER2 antibody or a mixture of such antibodymolecules and suitable excipients as outlined below and may additionallycomprise a soluble hyaluronidase glycoprotein either as a combinedformulation or as a separate formulation for co-administration.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows stability of the formulations A to F (see Table 1 below)after 8 weeks with respect to Low Molecular Weight (LMW) Speciesdetected by Size Exclusion-HPLC. As shown in this figure the PS20formulations A, C and E showed a slightly better stability than the PS80formulations B, D and F upon storage at 30° C.

FIG. 2 shows stability of the formulations A to F (see Table 1 below)after 8 weeks with respect to High Molecular Weight (HMW) Speciesdetected by Size Exclusion-HPLC. As shown in this figure theformulations A and B that contain trehalose without the addition ofsodium chloride show a smaller increase of HMWs after 8 weeks storagetime.

FIG. 3 shows stability after 8 weeks with respect to turbidity. As shownin this figure trehalose containing formulations A and B show a lowturbidity; whereas NaCl containing formulations C to F show a muchhigher turbidity. Formulations E and F containing trehalose as well asNaCl showed an intermediate turbidity. No significant increase wasobserved upon storage for 8 weeks.

FIG. 4 shows viscosity of liquid formulations A to F (see Table 1 below)as measured by using plate-cone viscosimetry at ambient temperature. Allformulations are in a low viscosity range that allow for subcutaneousinjection.

DETAILED DESCRIPTION OF THE INVENTION

The highly concentrated, stable pharmaceutical formulation of apharmaceutically active anti-HER2 antibody formulation of the presentinvention may be provided in liquid form or may be provided inlyophilized form. In accordance with the teachings in WO 97/04801 theantibody concentration in the reconstituted formulation can be increasedby reconstitution of a lyophilized formulation to provide a proteinconcentration in the reconstituted formulation which is about 2-40 timesgreater than the protein concentration in the mixture before thelyophilization step.

The anti-HER2 antibody concentration is 100 to 150 mg/ml, e.g. 120±18mg/ml, about 110 mg/ml, about 120 mg/ml or about 130 mg/ml.

The concentration of the buffering agent providing a pH of 5.5±2.0 is 1to 50 mM, e.g. 10 to 30 mM or about 20 mM. Various buffering agents areknown to the person skilled in the art as outlined further below. Thebuffering agent can be a histidine buffer, e.g. L-histidine/HCl. In aparticular embodiment the pH of the L-histidine/HCl buffer is about 5.5or about 6.0.

The stabilizer (used synonymously with the term “stabilizing agent” inthe present patent description) is e.g. a carbohydrate or saccharide ora sugar admitted by the authorities as a suitable additive or excipientin pharmaceutical formulations, e.g. α,α-trehalose dihydrate or sucrose.The concentration of the stabilizer is 15 to 250 mM, or 150 to 250 mM,or about 210 mM. The formulation may contain a secondary stabilizer,whereby this secondary stabilizer can be methionine, e.g in aconcentration of 5 to 25 mM or in a concentration of 5 to 15 mM (e.g.methionine in a concentration of about 5 mM, about 10 mM or about 15mM).

Suitable examples of pharmaceutically acceptable surfactants includepolyoxyethylensorbitan fatty acid esters (Tween),polyethylene-polypropylene glycols, polyoxyethylene-stearates,polyoxyethylene alkyl ethers, e.g. polyoxyethylene monolauryl ether,alkylphenylpolyoxyethylene ethers (Triton-X),polyoxyethylene-polyoxypropylene copolymer (Poloxamer, Pluronic), andsodium dodecyl sulphate (SDS). Most suitablepolyoxyethylenesorbitan-fatty acid esters are polysorbate 20, (soldunder the trademark Tween 20™) and polysorbate 80 (sold under thetrademark Tween 80™). Most suitable polyethylene-polypropylenecopolymers are those sold under the names Pluronic® F68 or Poloxamer188™. Most suitable polyoxyethylene alkyl ethers are those sold underthe trademark Brij™. Most suitable alkylphenolpolyoxyethylene ethers aresold under the trade name Triton-X. The nonionic surfactant can be apolysorbate, e.g. selected from the group of polysorbate 20, polysorbate80 and polyethylene-polypropylene copolymer. The concentration of thenonionic surfactant is 0.01 to 0.1% (w/v), or 0.01 to 0.08% (w/v), or0.025 to 0.075% (w/v), or more particularly about 0.02, 0.04 or 0.06%(w/v).

The concentration of the hyaluronidase enzyme depends on the actualhyaluronidase enzyme used in the preparation of the formulation inaccordance with the invention. An effective amount of the hyaluronidaseenzyme can easily be determined by the person skilled in the art basedon the disclosure further below. It should be provided in sufficientamount so that an increase in the dispersion and absorption of theco-administered anti-HER2 antibody is possible. The minimal amount ofthe hyaluronidase enzyme is >150 U/ml. More particularly the effectiveamount of the hyaluronidase enzyme is about 1'000 to 16'000 U/ml,whereby the said amount corresponds to about 0.01 mg to 0.16 mg proteinbased on an assumed specific activity of 100'000 U/mg. Alternatively theconcentration of the hyaluronidase enzyme is about 1′500 to 12'000 U/ml,or more particularly about 2'000 U/ml or about 12'000 U/ml. The amountsspecified hereinbefore correspond to the amount of hyaluronidase enzymeinitially added to the formulation. As evidenced in the exampleformulations the hyaluronidase enzyme concentrations measured in thefinal formulation may vary within a certain range. Thus, e.g. theactually measured hyaluronidase enzyme (HE) concentration measured justafter adding 12'000 U/ml of enzyme showed variations between 12355 U/mlto 15178 U/ml (see Table 1 Formulations A to F and Table 3 FormulationH). The hyaluronidase enzyme is present either as a combined finalformulation or for use for co-administration, e.g. as a co-formulationas further outlined below. The important issue for the formulation inaccordance with the present invention is that at the time it is readyfor use and/or is injected it has the composition as set out in theappended claims. The ratio (w/w) of the hyaluronidase enzyme to theanti-HER2 antibody is in the range of 1:1'000 to 1:8'000, or in therange of 1:4'000 to 1:5'000 or about 1:6'000.

The hyaluronidase enzyme may be derived from animals, human samples ormanufactured based on the recombinant DNA technology as describedfurther below.

In some embodiments the highly concentrated, stable pharmaceuticalanti-HER2 antibody formulations in accordance with the present inventionhave one of the following compositions:

-   -   a) 100 to 150 mg/ml anti-HER2 antibody, e.g. selected from the        group of Trastuzumab, Pertuzumab and T-DM1; 1 to 50 mM of a        histidine buffer, e.g. L-histidine/HCl at a pH of about 5.5; 15        to 250 mM of a stabilizer which is e.g. α,α-trehalose dihydrate,        and optionally methionine as a second stabilizer at a        concentration of 5 to 25 mM; about 0.01 to 0.08% of a nonionic        surfactant; and >150 to 16'000 U/ml, more particularly 1'000 to        16'000 U/ml of a hyaluronidase enzyme such as e.g. rHuPH20, e.g.        at a concentration of about 2'000 U/ml or about 12'000 U/ml.    -   b) 120±18 mg/ml anti-HER2 antibody, e.g. selected from the group        of Trastuzumab, Pertuzumab and T-DM1; 10 to 30 mM, or about 20        mM of a histidine buffer such as e.g. L-histidine/HCl at a pH of        about 5.5; 150 to 250 mM or about 210 mM of a stabilizer, which        is e.g. α,α-trehalose dihydrate, and optionally methionine as a        second stabilizer at a concentration of 5 to 25 mM, or 5 to 15        mM, or about 10 mM; about 0.01 to 0.08% of a nonionic        surfactant; and 1'000 to 16'000 U/ml, or 1'500 to 12'000 U/ml,        about 2'000 U/ml or about 12'000 U/ml of a hyaluronidase enzyme        such as e.g. rHuPH20.    -   c) About 120 mg/ml anti-HER2 antibody, e.g. selected from the        group of Trastuzumab, Pertuzumab and T-DM1; 10 to 30 mM, or        about 20 mM of a histidine buffer, such as e.g. L-histidine/HCl        at a pH of about 5.5; 150 to 250 mM, e.g. about 210 mM of a        stabilizer, which is e.g. α,α-trehalose dihydrate, and        optionally methionine as a second stabilizer at a concentration        of 5 to 25 mM, or 5 to 15 mM, or about 10 mM; about 0.01 to        0.08% of a nonionic surfactant; and 1'000 to 16'000 U/ml, or        1'500 to 12'000 U/ml, or more particularly about 2'000 U/ml or        about 12'000 U/ml of a hyaluronidase enzyme such as e.g.        rHuPH20.    -   d) About 120 mg/ml anti-HER2 antibody, e.g. selected from the        group of Trastuzumab, Pertuzumab and T-DM1; about 20 mM of a        histidine buffer such as e.g. L-histidine/HCl at a pH of about        5.5; about 210 mM α,α-trehalose dihydrate, and optionally about        10 mM methionine as a second stabilizer; 0.04 or 0.06% of        polysorbate 20; and about 12'000 U/ml of a hyaluronidase enzyme        such as rHuPH20; and particularly the Formulation A specified        below.    -   e) About 120 mg/ml anti-HER2 antibody, e.g. selected from the        group of Trastuzumab, Pertuzumab and T-DM1; about 20 mM of a        histidine buffer such as e.g. L-histidine/HCl at a pH of about        5.5; about 210 mM α,α-trehalose dihydrate, and optionally 10 mM        methionine as a second stabilizer; 0.04 or 0.06% of polysorbate        20; and about 2'000 U/ml of a hyaluronidase enzyme such as        rHuPH20; and particularly the Formulation X specified below.    -   f) A lyophilized formulation comprising 120 mg/ml anti-HER2        antibody, e.g. selected from the group of Trastuzumab,        Pertuzumab and T-DM1; 20 mM of a histidine buffer such as e.g.        L-histidine/HCl at a pH of about 5.5; 210 mM of α,α-trehalose        dihydrate and optionally 10 mM methionine as a second        stabilizer; about 0.04 to 0.06% of a nonionic surfactant; and        particularly Formulation Y specified below. These formulations        can be reconstituted with 1'000 to 16'000 U/ml, or 1'500 to        12'000 U/ml, or more particularly about 2'000 U/ml or about        12'000 U/ml of a hyaluronidase enzyme such as e.g. rHuPH20.

In another embodiment the highly concentrated, stable pharmaceuticalanti-HER2 antibody formulations in accordance with the present inventionhave one of the compositions specified in Table 1, 3 and 4, whereby theformulations C, D, E and F are less preferred because of less desiredproperties as outlined in the Examples and in Table 1.

It has been proposed to facilitate the subcutaneous injection oftherapeutic proteins and antibodies by using small amounts of solublehyaluronidase glycoproteins (sHASEGPs); see WO2006/091871. It has beenshown that the addition of such soluble hyaluronidase glycoproteins(either as a combined formulation or by co-administration) facilitatesthe administration of therapeutic drug into the hypodermis. By rapidlydepolymerizing hyaluronan HA in the extracellular space sHASEGP reducesthe viscosity of the interstitium, thereby increasing hydraulicconductance and allowing for larger volumes to be administered safelyand comfortably into the subcutaneous tissue. The increased hydraulicconductance induced by sHASEGP through reduced interstitial viscosityallows for greater dispersion, potentially increasing the systemicbioavailability of SC administered therapeutic drug.

The highly concentrated, stable pharmaceutical formulations of thepresent invention comprising a soluble hyaluronidase glycoprotein aretherefore particularly suited for subcutaneous injection. It is clearlyunderstood by the person skilled in the art that such a formulationcomprising an anti-HER2 antibody and a soluble hyaluronidaseglycoprotein can be provided for administration in form of one singlecombined formulation or alternatively in form of two separateformulations which can be mixed just prior to the subcutaneousinjection. Alternatively the anti-HER2 antibody and the solublehyaluronidase glycoprotein can be administered as separate injections atdifferent sites of the body, preferably at sites which are immediatelyadjacent to each other. It is also possible to inject the therapeuticagents present in the formulation in accordance with the presentinvention as consecutive injections, e.g. first the solublehyaluronidase glycoprotein followed by the injection of the anti-HER2antibody formulation. These injections can also be performed in thereversed order, viz. by first injecting the anti-HER2 antibodyformulation followed by injecting the soluble hyaluronidaseglycoprotein. In case the anti-HER2 antibody and the solublehyaluronidase glycoprotein are administered as separate injections, oneor both of the proteins have to be provided with the buffering agent,the stabilizer(s) and the nonionic surfactant in the concentrations asspecified in the appended claims but excluding the hyaluronidase enzyme.The hyaluronidase enzyme can then be provided e.g. in a L-histidine/HClbuffer at pH of about 6.5, 100 to 150 mM NaCl and 0.01 to 0.1% (w/v)polysorbate 20 or polysorbate 80. In particular the hyaluronidase enzymeis provided in 20 mM L-histidine/HCl buffer at pH 6.5, 130 mM NaCl,0.05% (w/v) polysorbate 80 as specifically exemplified in Formulation Gof Table 1 below.

As noted above the soluble hyaluronidase glycoprotein may be consideredto be a further excipient in the anti-HER2 formulation. The solublehyaluronidase glycoprotein may be added to the anti-HER2 formulation atthe time of manufacturing the anti-HER2 formulation or may be addedshortly before the injection. Alternatively the soluble hyaluronidaseglycoprotein may be provided as a separate injection. In the latter casethe soluble hyaluronidase glycoprotein may be provided in a separatevial either in lyophilized form which must be reconstituted withsuitable diluents before the subcutaneous injection takes place, or maybe provided as a liquid formulation by the manufacturer. The anti-HER2formulation and the soluble hyaluronidase glycoprotein may be procuredas separate entities or may also be provided as kits comprising bothinjection components and suitable instructions for their subcutaneousadministration. Suitable instructions for the reconstitution and/oradministration of one or both of the formulations may also be provided.

A variety of anti-HER2 antibodies are known in the prior art. Suchantibodies are preferably monoclonal antibodies. They may either beso-called chimaeric antibodies, humanized antibodies or fully humanantibodies. They may either be full length anti-HER2 antibodies;anti-HER2 antibody fragments having the same biological activity;including amino acid sequence variants and/or glycosylation variants ofsuch antibodies or antibody fragments. Examples of humanized anti-HER2antibodies are known under the INN names Trastuzumab and Pertuzumab.Another suitable anti-HER2 antibody is T-DM1, which is an antibody-toxinconjugate consisting of huMAb4D5-8 (HERCEPTIN™) and a maytansinoide(viz. DM1=N^(2′)-deacetyl-N^(2′)-(3-mercapto-1-oxopropyl)-maytansine; ahighly potent antimicrotubule agent) which conjugate (with a MCC linker)is currently under development for metastatic breast cancer. Other HER2antibodies with various properties have been described in Tagliabue etal., Int. J. Cancer, 47:933-937 (1991); McKenzie et al., Oncogene,4:543-548 (1989); Cancer Res., 51:5361-5369 (1991); Bacus et al.,Molecular Carcinogenesis, 3:350-362 (1990); Stancovski et al., PNAS(USA), 88:8691-8695 (1991); Bacus et al., Cancer Research, 52:2580-2589(1992); Xu et al., Int. J. Cancer, 53:401-408 (1993); WO94/00136;Kasprzyk et al., Cancer Research, 52:2771-2776 (1992); Hancock et al.,Cancer Res., 51:4575-4580 (1991); Shawver et al., Cancer Res.,54:1367-1373 (1994); Arteaga et al., Cancer Res., 54:3758-3765 (1994);Harwerth et al., J. Biol. Chem., 267:15160-15167 (1992); U.S. Pat. No.5,783,186; and Klapper et al., Oncogene, 14:2099-2109 (1997). The mostsuccessful therapeutic anti-HER2 antibody is Trastuzumab sold byGenentech Inc. and F. Hoffmann-La Roche Ltd under the trade nameHERCEPTIN™. Further details on the HER2 antigen and antibodies directedthereto are described in many patent and non-patent publications (for asuitable overview see U.S. Pat. No. 5,821,337 and WO 2006/044908).

The anti-HER2 antibody is e.g. selected from the group of Trastuzumab,Pertuzumab and T-DM1 and may also consist of a mixture of anti-HER2antibodies such as e.g. Trastuzumab and Pertuzumab or T-DM1 andPertuzumab. It has been found that the combination of Pertuzumab andTrastuzumab is active and well tolerated in patients with metastaticHER2-positive breast cancer who had experienced progression during priortrastuzumab therapy [see e.g. Baselga, J. et al., Journal of Clin.Oncol. Vol 28 (7) 2010: pp. 1138-1144]. The formulation in accordancewith the present invention is exemplified herein with the anti-HER2antibody Trastuzumab. The terms “Trastuzumab”, “Pertuzumab” and “T-DM1”encompass all corresponding anti-HER2 antibodies that full-fill therequirements necessary for obtaining a marketing authorization as anidentical or biosimilar product in a country or territory selected fromthe group of countries consisting of the USA, Europe and Japan.Trastuzumab has the CDR regions defined in EP-B-590058. Pertuzumab hasthe CDR regions defined in WO 01/00245. The activity of Trastuzumab inthe BT-474 antiproliferation assay [Nahta, R. et al., “TheHER-2-targeting antibodies Trastuzumab and Pertuzumab synergisticallyinhibit the survival of breast cancer cells”, Cancer Res. 2004;64:2343.2346] has been found to be between 0.7-1.3×10⁴ U/mg. T-DM1 isdescribed in WO 2005/117986.

HERCEPTIN™ (Trastuzumab) has been approved in the EU for the treatmentof patients with metastatic breast cancer (MBC) who have tumors thatoverexpress HER2 as follows:

-   -   As monotherapy for the treatment of patients who have received        at least two chemotherapy regimens for their metastatic disease.        Prior chemotherapy must have included at least an anthracycline        and a taxane unless patients are unsuitable for these        treatments. Hormone receptor-positive patients must also have        failed hormonal therapy, unless patients are unsuitable for        these treatments.    -   In combination with paclitaxel for the treatment of those        patients who have not received chemotherapy for their metastatic        disease and for whom an anthracycline is not suitable.    -   In combination with docetaxel for the treatment of those        patients who have not received chemotherapy for their metastatic        disease.    -   In combination with an aromatase inhibitor for the treatment of        postmenopausal patients with hormone receptor-positive MBC not        previously treated with Trastuzumab.

Trastuzumab has also been approved in the EU for the treatment ofpatients with MBC who have tumors that overexpress HER2 for thetreatment of patients with HER2-positive early-stage breast cancer (EBC)following surgery, chemotherapy (neoadjuvant or adjuvant) andradiotherapy (if applicable).

Moreover Trastuzumab is currently being developed for the treatment ofgastric cancer.

Two dosing regimens are currently approved for Trastuzumab (Table 1);once weekly (q1w) and every 3 weeks (q3w) for both metastatic breastcancer (MBC) and early breast cancer (EBC). In the q1w dosing regime theloading dose is 4 mg/kg followed by subsequent dose at 2 mg/kg. In theq3w dosing regime the loading dose is 8 mg/kg followed by subsequentdose at 6 mg/kg.

As noted above HERCEPTIN™ (Trastuzumab) for intravenous administrationis currently sold in lyophilized form in vials. In the formulation soldin Europe each vial contains the dry residue obtained afterlyophilization of a filling volume of 6.25 ml of a sterile aqueoussolution containing the following components: 150 mg Trastuzumab(effective 156.3 mg to ensure that the nominal quantity of 150 mg can bewithdrawn from the final product after reconstitution), 3.50 mgL-histidine hydrochloride, 2.25 mg L-histidine, 141.9 mg α,α-trehalosedihydrate, 0.63 mg Polysorbate 20. The dissolved lyophilisate containsabout 24 mg/ml Trastuzumab, 5 mM L-histidine/HCl pH 6.0, 60 mMα,α-trehalose dihydrate, 0.01% polysorbate 20. The solution is thenadded to the infusion solution and then the infusion is administered tothe patient over 90 minutes (subsequent infusions can be given over 30minutes in MBC, if well tolerated).

A number of a soluble hyaluronidase glycoprotein are known in the priorart. In order to further define the function, the mechanism of actionand the properties of such soluble hyaluronidase glycoproteins thefollowing background information is provided.

The SC (hypodermal) interstitial matrix is comprised of a network offibrous proteins embedded within a viscoelastic gel ofglycosaminoglycans. Hyaluronan (HA), a non-sulfated repeating lineardisaccharide, is the prominent glycosaminoglycan of the SC tissue. HA issecreted into the interstitium by fibroblasts as a high molecularweight, megadalton viscous polymer that is subsequently degradedlocally, in the lymph, and in the liver, through the action of lysosomalhyaluronidases and exoglycosidases. Approximately 50% of the hyaluronanin the body is produced by the SC tissue, where it is found atapproximately 0.8 mg/gm wet weight tissue [Aukland K. and Reed R.,supra]. It is estimated that the average 70 kg adult contains 15 gramsof HA, of which 30 percent is turned over (synthesized and degraded)daily [Laurent L. B., et al., “Catabolism of hyaluronan in rabbit skintakes place locally, in lymph nodes and liver”, Exp. Physiol. 1991; 76:695-703]. As a major constituent of the gel-like component of thehypodermal matrix, HA contributes significantly to its viscosity.

Glycosaminoglycans (GAGs) are complex linear polysaccharides of theextracellular matrix (ECM). GAGs are characterized by repeatingdisaccharide structures of an N-substituted hexosamine and an uronicacid (in the case of hyaluronan (HA), chondroitin sulfate (CS),chondroitin (C), dermatan sulfate (DS), heparan sulfate (HS), andheparin (H)), or a galactose (in the case of keratan sulfate (KS)).Except for HA, all exist covalently bound to core proteins. The GAGswith their core proteins are structurally referred to as proteoglycans(PGs).

Hyaluronan (HA) is found in mammals predominantly in connective tissues,skin, cartilage, and in synovial fluid. Hyaluronan is also the mainconstituent of the vitreous of the eye. In connective tissue, the waterof hydration associated with hyaluronan creates hydrated matricesbetween tissues. Hyaluronan plays a key role in biological phenomenaassociated with cell motility including rapid development, regeneration,repair, embryogenesis, embryological development, wound healing,angiogenesis, and tumorigenesis (Toole, Cell Biol. Extracell. Matrix,Hay (ed), Plenum Press, New York, 1991; pp. 1384-1386; Bertrand et al.,Int. J. Cancer 1992; 52:1-6; Knudson et al., FASEB J. 1993;7:1233-1241]. In addition, hyaluronan levels correlate with tumoraggressiveness [Ozello et al., Cancer Res. 1960; 20:600-604; Takeuchi etal., Cancer Res. 1976; 36:2133-2139; Kimata et al., Cancer Res. 1983;43:1347-1354].

HA is found in the extracellular matrix of many cells, especially insoft connective tissues. HA has been assigned various physiologicalfunctions, such as in water and plasma protein homeostasis [Laurent T.C. et al., FASEB J., 1992; 6: 2397-2404]. HA production increases inproliferating cells and may play a role in mitosis. It has also beenimplicated in locomotion and cell migration. HA seems to play importantroles in cell regulation, development, and differentiation [Laurent etal., supra].

HA has widely been used in clinical medicine. Its tissue protective andrheological properties have proved useful in ophthalmic surgery (e.g. toprotect the corneal endothelium during cataract surgery). Serum HA isdiagnostic of liver disease and various inflammatory conditions, such asrheumatoid arthritis. Interstitial edema caused by accumulation of HAmay cause dysfunction in various organs [Laurent et al., supra].

Hyaluronan protein interactions also are involved in the structure ofthe extracellular matrix or “ground substance”.

Hyaluronidases are a group of generally neutral- or acid-active enzymesfound throughout the animal kingdom. Hyaluronidases vary with respect tosubstrate specificity, and mechanism of action (WO 2004/078140). Thereare three general classes of hyaluronidases:

1. Mammalian-type hyaluronidases, (EC 3.2.1.35) which areendo-beta-N-acetylhexosaminidases with tetrasaccharides andhexasaccharides as the major end products. They have both hydrolytic andtransglycosidase activities, and can degrade hyaluronan and chondroitinsulfates (CS), generally C4-S and C6-S.2. Bacterial hyaluronidases (EC 4.2.99.1) degrade hyaluronan and, and tovarious extents, CS and DS. They are endo-beta-N-acetylhexosaminidasesthat operate by a beta elimination reaction that yields primarilydisaccharide end products.3. Hyaluronidases (EC 3.2.1.36) from leeches, other parasites, andcrustaceans are endo-beta-glucuronidases that generate tetrasaccharideand hexasaccharide end products through hydrolysis of the beta 1-3linkage.

Mammalian hyaluronidases can be further divided into two groups:neutral-active and acid-active enzymes. There are six hyaluronidase-likegenes in the human genome, HYAL1, HYAL2, HYAL3, HYAL4, HYALP1 andPH20/SPAM1. HYALP1 is a pseudogene, and HYAL3 has not been shown topossess enzyme activity toward any known substrates. HYAL4 is achondroitinase and exhibits little activity towards hyaluronan. HYAL1 isthe prototypical acid-active enzyme and PH20 is the prototypicalneutral-active enzyme. Acid-active hyaluronidases, such as HYAL1 andHYAL2 generally lack catalytic activity at neutral pH (i.e. pH 7). Forexample, HYAL1 has little catalytic activity in vitro over pH 4.5 [FrostI. G. and Stern, R., “A microtiter-based assay for hyaluronidaseactivity not requiring specialized reagents”, Anal. Biochemistry, 1997;251:263-269]. HYAL2 is an acid-active enzyme with a very low specificactivity in vitro.

The hyaluronidase-like enzymes can also be characterized by those whichare generally locked to the plasma membrane via a glycosylphosphatidylinositol anchor such as human HYAL2 and human PH20[Danilkovitch-Miagkova et al., Proc. Natl. Acad. Sci. USA, 2003;100(8):4580-4585; Phelps et al., Science 1988; 240(4860): 1780-1782],and those which are generally soluble such as human HYAL1 [Frost, I. G.et al., “Purification, cloning, and expression of human plasmahyaluronidase”, Biochem. Biophys. Res. Commun. 1997; 236(1):10-15].However, there are variations from species to species: bovine PH20 forexample is very loosely attached to the plasma membrane and is notanchored via a phospholipase sensitive anchor [Lalancette et al., Biol.Reprod., 2001; 65(2):628-36]. This unique feature of bovinehyaluronidase has permitted the use of the soluble bovine testeshyaluronidase enzyme as an extract for clinical use (Wydase™, Hyalase™).Other PH20 species are lipid anchored enzymes that are generally notsoluble without the use of detergents or lipases. For example, humanPH20 is anchored to the plasma membrane via a GPI anchor. Attempts tomake human PH20 DNA constructs that would not introduce a lipid anchorinto the polypeptide resulted in either a catalytically inactive enzyme,or an insoluble enzyme [Arming et al., Eur. J. Biochem., 1997;247(3):810-4]. Naturally occurring macaque sperm hyaluronidase is foundin both a soluble and membrane bound form. While the 64 kDa membranebound form possesses enzyme activity at pH 7.0, the 54 kDa form is onlyactive at pH 4.0 [Cherr et al., Dev. Biol., 1996; 10; 175(1): 142-53].Thus, soluble forms of PH20 are often lacking enzyme activity underneutral conditions.

As noted above and in accordance with the teachings in WO2006/091871small amounts of soluble hyaluronidase glycoproteins (sHASEGPs) can beintroduced into a formulation in order to facilitate the administrationof therapeutic drug into the hypodermis. By rapidly depolymerizing HA inthe extracellular space sHASEGP reduces the viscosity of theinterstitium, thereby increasing hydraulic conductance and allowing forlarger volumes to be administered safely and comfortably into the SCtissue. The increased hydraulic conductance induced by sHASEGP throughreduced interstitial viscosity allows for greater dispersion,potentially increasing the systemic bioavailability of SC administeredtherapeutic drug.

When injected in the hypodermis, the depolymerization of HA by sHASEGPis localized to the injection site in the SC tissue. Experimentalevidence shows that the sHASEGP is inactivated locally in theinterstitial space with a half life of 13 to 20 minutes in mice, withoutdetectable systemic absorption in blood following single intravenousdose in CD-1 mice. Within the vascular compartment sHASEGP demonstratesa half life of 2.3 and 5 minutes in mice and Cynomolgus monkeys,respectively, with doses up to 0.5 mg/kg. The rapid clearance ofsHASEGP, combined with the continual synthesis of the HA substrate inthe SC tissue, results in a transient and locally-active permeationenhancement for other co-injected molecules, the effects of which arefully reversible within 24 to 48 hours post administration [Bywaters G.L., et al., “Reconstitution of the dermal barrier to dye spread afterHyaluronidase injection”, Br. Med. J., 1951; 2 (4741): 1178-1183].

In addition to its effects on local fluid dispersion, sHASEGP also actsas absorption enhancer. Macromolecules greater than 16 kilodaltons (kDa)are largely excluded from absorption through the capillaries viadiffusion and are mostly absorbed via the draining lymph nodes. Asubcutaneously administered macromolecule such as e.g. a therapeuticantibody (molecular weight approximately 150 kDa) must thereforetraverse the interstitial matrix before reaching the draining lymphaticsfor subsequent absorption into the vascular compartment. By increasinglocal dispersion, sHASEGP increases the rate (Ka) of absorption of manymacromolecules. This leads to increased peak blood levels (C_(max)) andpotentially to increased bioavailability relative to SC administrationin the absence of sHASEGP [Bookbinder L. H., et al., “A recombinanthuman enzyme for enhanced interstitial transport of therapeutics”, J.Control. Release 2006; 114: 230-241].

Hyaluronidase products of animal origin have been used clinically forover 60 years, primarily to increase the dispersion and absorption ofother co-administered drugs and for hypodermoclysis (SCinjection/infusion of fluid in large volume) [Frost G. I., “Recombinanthuman hyaluronidase (rHuPH20): an enabling platform for subcutaneousdrug and fluid administration”, Expert Opinion on Drug Delivery, 2007;4: 427-440]. The details on the mechanism of action of hyaluronidaseshave been described in detail in the following publications:Duran-Reynolds F., “A spreading factor in certain snake venoms and itsrelation to their mode of action”, CR Soc Biol Paris, 1938; 69-81; ChainE., “A mucolytic enzyme in testes extracts”, Nature 1939; 977-978;Weissmann B., “The transglycosylative action of testicularhyaluronidase”, J. Biol. Chem., 1955; 216: 783-94; Tammi, R., Saamanen,A. M., Maibach, H. I., Tammi M., “Degradation of newly synthesized highmolecular mass hyaluronan in the epidermal and dermal compartments ofhuman skin in organ culture”, J. Invest. Dermatol. 1991; 97:126-130;Laurent, U. B. G., Dahl, L. B., Reed, R. K., “Catabolism of hyaluronanin rabbit skin takes place locally, in lymph nodes and liver”, Exp.Physiol. 1991; 76: 695-703; Laurent, T. C. and Fraser, J. R. E.,“Degradation of Bioactive Substances: Physiology and Pathophysiology”,Henriksen, J. H. (Ed) CRC Press, Boca Raton, Fla.; 1991. pp. 249-265;Harris, E. N., et al., “Endocytic function, glycosaminoglycanspecificity, and antibody sensitivity of the recombinant human 190-kDahyaluronan receptor for endocytosis (HARE)”, J. Biol. Chem. 2004;279:36201-36209; Frost, G. I., “Recombinant human hyaluronidase(rHuPH20): an enabling platform for subcutaneous drug and fluidadministration”, Expert Opinion on Drug Delivery, 2007; 4: 427-440.Hyaluronidase products approved in EU countries include Hylase® “Dessau”and Hyalase®. Hyaluronidase products of animal origin approved in the USinclude Vitrase™, Hydase™, and Amphadase™.

The safety and efficacy of hyaluronidase products have been widelyestablished. The most significant safety risk identified ishypersensitivity and/or allergenicity, which is thought to be related tothe lack of purity of the animal-derived preparations [Frost, G. I.,“Recombinant human hyaluronidase (rHuPH20): an enabling platform forsubcutaneous drug and fluid administration”, Expert Opinion on DrugDelivery, 2007; 4: 427-440]. It should be noted that there aredifferences with respect to the approved dosages of animal-derivedhyaluronidases between the UK, Germany and the US. In the UK, the usualdose as an adjuvant to subcutaneous or intramuscular injection is 1500units, added directly to the injection. In the US, the usual dose usedfor this purpose is 150 units. In hypodermoclysis, hyaluronidase is usedto aid the subcutaneous administration of relatively large volumes offluids. In the UK, 1500 units of hyaluronidase are generally given witheach 500 to 1000 ml of fluid for subcutaneous use. In the US, 150 unitsare considered adequate for each liter of hypodermoclysis solution. InGermany, 150 to 300 units are considered adequate for this purpose. Inthe UK, the diffusion of local anesthetics is accelerated by theaddition of 1500 units. In Germany and the US 150 units are consideredadequate for this purpose. The dosage differences notwithstanding (thedosage in the UK is ten times higher than in the US), no apparentdifferences in the safety profiles of animal-derived hyaluronidaseproducts marketed in the US and UK, respectively, have been reported.

On Dec. 2, 2005, Halozyme Therapeutics Inc. received approval from theFDA for an injectable formulation of the recombinant humanhyaluronidase, rHuPH20 (HYLENEX™). The FDA approved HYLENEX™ at a doseof 150 units for SC administration of the following indications:

-   -   as an adjuvant to increase the absorption and dispersion of        other injected drugs    -   for hypodermoclysis    -   as an adjunct in SC urography for improving resorption of        radiopaque agents.

As part of that regulatory review it was established that rHuPH20possesses the same properties of enhancing the dispersion and absorptionof other injected drugs as the previously approved animal-derivedhyaluronidase preparations, but with an improved safety profile. Inparticular, the use of recombinant human hyaluronidase (rHuPH20)compared with animal-derived hyaluronidases minimizes the potential riskof contamination with animal pathogens and transmissible spongiformencephalopathies.

Soluble Hyaloronidase glycoproteins (sHASEGP), a process for preparingthe same and their use in pharmaceutical compositions have beendescribed in WO 2004/078140. The use of soluble Hyaloronidaseglycoproteins in combination with a variety of exemplary antibodies,such as e.g. Trastuzumab, has been mentioned in WO 2006/091871.

The detailed experimental work as outlined further below has shown thatthe formulations of the present invention surprisingly have favorablestorage stability and fulfill all necessary requirements for approval bythe health authorities.

The hyaluronidase enzyme in the formulations of the present inventionenhances the delivery of the anti-HER2 antibody to the systemiccirculation, e.g. by increasing the absorption of the active substance(it acts as a permeation enhancer). The hyaluronidase enzyme alsoincreases the delivery of the therapeutic anti-HER2 antibody into thesystemic circulation via the subcutaneous application route by thereversible hydrolyzation of hyaluronan, an extracellular component ofthe SC interstitial tissue. The hydrolysis of hyaluronan in thehypodermis temporarily opens channels in the interstitial space of theSC tissue and thereby improves the delivery of the therapeutic anti-HER2antibody into the systemic circulation. In addition, the administrationshows reduced pain in humans and less volume-derived swelling of the SCtissue.

Hyaluronidase, when administered locally has its entire effect locally.In other word hyaluronidase is inactivated and metabolized locally inminutes and has not been noted to have systemic or long term effects.The rapid inactivation of hyaluronidase within minutes when it entersthe blood stream precludes a realistic ability to perform comparablebiodistribution studies between different hyaluronidase products. Thisproperty also minimizes any potential systemic safety concerns becausethe hyaluronidase product cannot act at distant sites.

The unifying feature of all hyaluronidase enzymes is their ability todepolymerize hyaluronan, regardless of differences in chemicalstructure, in species source, in tissue sources, or in the batches ofdrug product sourced from the same species and tissue. They are unusualin that their activity is the same (except for potency) in spite ofhaving different structures.

The hyaluronidase enzyme excipient in accordance with the formulation ofthe present invention is characterized by having no adverse effect onthe molecular integrity of the anti-HER2 antibody in the stablepharmaceutical formulation described herein. Furthermore, thehyaluronidase enzyme merely modifies the delivery of the anti-HER2antibody to the systemic circulation but does not possess any propertiesthat could provide or contribute to the therapeutic effects ofsystemically absorbed anti-HER2 antibody. The hyaluronidase enzyme isnot systemically bioavailable and does not adversely affect themolecular integrity of the anti-HER2 antibody at the recommended storageconditions of the stable pharmaceutical formulation in accordance withthe invention. It is therefore to be considered as an excipient in theanti-HER2 antibody formulation in accordance with this invention. As itexerts no therapeutic effect it represents a constituent of thepharmaceutical form apart from the therapeutically active anti-HER2antibody.

A number of suitable hyaluronidase enzymes in accordance with thepresent invention are known from the prior art. The preferred enzyme isa human hyaluronidase enzyme, most preferably the enzyme known asrHuPH20. rHuPH20 is a member of the family of neutral and acid-activeβ-1,4 glycosyl hydrolases that depolymerize hyaluronan by the hydrolysisof the β-1,4 linkage between the C₁ position of N-acetyl glucosamine andthe C₄ position of glucuronic acid. Hyaluronan is a polysaccharide foundin the intracellular ground substance of connective tissue, such as thesubcutaneous interstitial tissue, and of certain specialized tissues,such as the umbilical cord and vitreous humor. The hydrolysis ofhyaluronan temporarily decreases the viscosity of the interstitialtissue and promotes the dispersion of injected fluids or of localizedtransudates or exudates, thus facilitating their absorption. The effectsof hyaluronidase are local and reversible with complete reconstitutionof the tissue hyaluronan occurring within 24 to 48 hours [Frost, G. I.,“Recombinant human hyaluronidase (rHuPH20): an enabling platform forsubcutaneous drug and fluid administration”, Expert Opinion on DrugDelivery, 2007; 4:427-440]. The increase in the permeability ofconnective tissue through the hydrolysis of hyaluronan correlates withthe efficacy of hyaluronidase for their capability to increase thedispersion and absorption of co-administered molecules.

The human genome contains several hyaluronidase genes. Only the PH20gene product possesses effective hyaluronidase activity underphysiologic extracellular conditions and acts as a spreading agent,whereas acid-active hyaluronidases do not have this property.

rHuPH20 is the first and only recombinant human hyaluronidase enzymecurrently available for therapeutic use. Naturally occurring human PH20protein has a lipid anchor attached to the carboxy terminal amino acidthat anchors it to the plasma membrane. The rHuPH20 enzyme developed byHalozyme is a truncated deletion variant that lacks such amino acids inthe carboxy terminus responsible for the lipid attachment. This givesrise to a soluble, neutral pH-active enzyme similar to the protein foundin bovine testes preparations. The rHuPH20 protein is synthesized with a35 amino acid signal peptide that is removed from the N-terminus duringthe process of secretion. The mature rHuPH20 protein contains anauthentic N-terminal amino acid sequence orthologous to that found insome bovine hyaluronidase preparations.

The PH20 hyaluronidases, including the animal derived PH20 andrecombinant human rHuPH20, depolymerize hyaluronan by the hydrolysis ofthe β-1,4 linkage between the C₁ position of N-acetyl glucosamine andthe C₄ position of glucuronic acid. The tetrasaccharide is the smallestdigestion product [Weissmann, B., “The transglycosylative action oftesticular hyaluronidase”, J. Biol. Chem., 1955; 216: 783-94]. ThisN-acetyl glucosamine/glucuronic acid structure is not found in N-linkedglycans of recombinant biological products and therefore rHuPH20 willnot affect the glycosylation of antibodies it is formulated with, suchas e.g. Trastuzumab. The rHuPH20 enzyme itself possesses six N-linkedglycans per molecule with core structures similar to that found inmonoclonal antibodies. As anticipated, these N-linked structures do notchange over time, confirming the lack of enzymatic activity of rHuPH20on these N-linked glycan structures. The short half life of rHuPH20 andthe constant synthesis of hyaluronan lead to a short and local action ofthe enzyme on tissues.

The hyaluronidase enzyme which is an excipient in the subcutaneousformulation in accordance with the present invention can be prepared byusing recombinant DNA technology. In this way it is ensured that thesame protein (identical amino acid sequence) is obtained all the timeand that an allergic reaction, e.g. caused by contaminating proteinsco-purified during extraction from a tissue, is avoided. Thehyaluronidase enzyme used in the formulation as exemplified herein is ahuman enzyme, viz. rHuPH20.

The amino acid sequence of rHuPH20 (HYLENEX™) is well known andavailable under CAS Registry No. 75971-58-7. The approximate molecularweight is 61 kDa.

Multiple structural and functional comparisons have been performedbetween naturally sourced mammalian hyaluronidase and PH-20 cDNA clonesfrom humans and other mammals. The PH-20 gene is the gene used for therecombinant product rHuPH20; however the recombinant drug product is a447 amino acid truncated version of the full protein encoded by thePH-20 gene. Structural similarities with respect to amino acid sequencesrarely exceed 60% in any comparison. Functional comparisons show thatthe activity of rHuPH20 is very similar to that of previously approvedhyaluronidase products. This information is consistent with the clinicalfindings during the past 50 years that regardless of the source of thehyaluronidase, the clinical safety and efficacy of units ofhyaluronidase are equivalent.

The use of rHuPH20 in the anti-HER2 antibody SC formulation inaccordance with the present invention allows the administration ofhigher volumes of drug product and to potentially enhance the absorptionof subcutaneously administered Trastuzumab into the systemiccirculation.

The osmolality of the stable pharmaceutical formulation in accordancewith the invention is 330±50 mOsm/kg.

The stable pharmaceutical formulation in accordance with the inventionis essentially free from visible (human eye inspection) particles. Thesub-visible particles (as measured by light obscuration) should fulfillthe following criteria:

-   -   maximum number of particles≥10 μm per vial->6000    -   maximum number of particles≥25 μm per vial->600

In a further aspect the present invention provides the use of aformulation for the preparation of a medicament useful for treating adisease or disorder amenable to treatment with an anti-HER2 antibodysuch as e.g. cancer or a non-malignant disease in a subject comprisingadministering the formulation described herein to a subject in an amounteffective to treat the said disease or disorder. The anti-HER2 antibodycan be co-administered concomitantly or sequentially with achemotherapeutic agent.

In a further aspect the present invention provides a method of treatinga disease or disorder which is amenable to treatment with an anti-HER2antibody (e.g. cancer or a non-malignant disease) in a subjectcomprising administering the formulation described herein to a subjectin an amount effective to treat the said disease or disorder. The canceror a non-malignant disease will generally involve HER2-expressing cells,such that the HER2 antibody in the therapeutic pharmaceutical SCformulation in accordance with the present invention is able to bind tothe affected cells. Various cancer or a non-malignant diseases that canbe treated with a formulation in accordance with the present inventionare listed below.

The stable pharmaceutical formulation of the pharmaceutically activeanti-HER2 antibody in accordance with the invention can be administeredas subcutaneous injection, whereby the administration is repeatedseveral times with time intervals of 3 weeks (q3w). The full volume ofthe injection fluid is in most cases administered within a time periodof 1 to 10 minutes, preferably 2 to 6 minutes, most preferably 3±1minutes. In adjuvant EBC patients and amongst patients with MBCreceiving Trastuzumab monotherapy, where no other intravenous (IV)chemotherapeutic agents are given, such subcutaneous administrationleads to increased patient convenience with the potential forself-administration at home. This leads to improved compliance andreduces/eliminates costs associated with IV administration (viz.,nursing costs for IV administration, rental of day-beds, patient traveletc). Subcutaneous administration in accordance with the presentinvention will most likely be associated with a reduced frequency and/orintensity of infusion-related reactions.

The addition of the hyaluronidase to the formulation allows increasingthe injection volume which can be safely and comfortably administeredsubcutaneously. Under normal circumstances the injection volume is 1 to15 ml. It has been observed that the administration of the formulationin accordance with the present invention increases the dispersion,absorption and the bioavailability of the therapeutic antibody. Largemolecules (i.e. >16 kDa) that are administered via the SC route arepreferentially absorbed into the vascular compartment through thedraining lymphatic fluids [Supersaxo, A., et al., “Effect of MolecularWeight on the Lymphatic Absorption of Water-Soluble Compounds FollowingSubcutaneous Administration”, 1990; 2:167-169; Swartz, M. A., “AdvancedDrug Delivery Review, The physiology of the lymphatic system”, 2001; 50:3-20]. The rate of introduction of these large molecules into thesystemic circulation is thus slowed relative to intravenous infusion,therefore potentially resulting in reduced frequency/intensity ofinfusion related reactions.

The production of the subcutaneous Trastuzumab formulation in accordancewith the invention requires high antibody concentrations (approx. 120mg/ml) in the final step of purification of the manufacturing process.Therefore an additional process step (ultrafiltration/diafiltration) isadded to the conventional manufacturing process of Trastuzumab. Inaccordance with the teachings in WO 97/04801 the highly concentrated,stable pharmaceutical anti-HER2 antibody formulation in accordance withthe present invention can also be provided as stabilized proteinformulation which can reconstituted with a suitable diluent to generatea high anti-HER2 antibody concentration reconstituted formulation.

The HER2 antibody SC formulation in accordance with this invention ismainly used to treat cancer. Whereby the terms “cancer” and “cancerous”refer to or describe the physiological condition in mammals that istypically characterized by unregulated cell growth. Examples of cancerinclude, but are not limited to, carcinoma, lymphoma, blastoma(including medulloblastoma and retinoblastoma), sarcoma (includingliposarcoma and synovial cell sarcoma), neuroendocrine tumors (includingcarcinoid tumors, gastrinoma and islet cell cancer), mesothelioma,schwannoma (including acoustic neuroma), meningioma, adenocarcinoma,melanoma, and leukemia or lymphoid malignancies. More particularexamples of such cancers include squamous cell cancer (e.g. epithelialsquamous cell cancer), lung cancer including small-cell lung cancer,non-small cell lung cancer, adenocarcinoma of the lung and squamouscarcinoma of the lung, cancer of the peritoneum, hepatocellular cancer,gastric or stomach cancer including gastrointestinal cancer, pancreaticcancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer,bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer,colorectal cancer, endometrial or uterine carcinoma, salivary glandcarcinoma, kidney or renal cancer, prostate cancer, vulval cancer,thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma,testicular cancer, esophageal cancer, tumors of the biliary tract, aswell as head and neck cancer.

The term “about” as used in the present patent specification is meant tospecify that the specific value provided may vary to a certain extent,such as e.g. means that variations in the range of ±10%, preferably ±5%,most preferably ±2% are included in the given value.

A cancer which “overexpresses” a HER receptor is one which hassignificantly higher levels of a HER receptor, such as HER2, at the cellsurface thereof, compared to a noncancerous cell of the same tissuetype. Such overexpression may be caused by gene amplification or byincreased transcription or translation. HER receptor overexpression maybe determined in a diagnostic or prognostic assay by evaluatingincreased levels of the HER protein present on the surface of a cell(e.g. via an immunohistochemistry assay; IHC). Alternatively, oradditionally, one may measure levels of HER-encoding nucleic acid in thecell, e.g. via fluorescent in situ hybridization (FISH; see WO98/45479),Southern blotting, or polymerase chain reaction (PCR) techniques, suchas real time quantitative PCR (RT-PCR). One may also study HER receptoroverexpression by measuring shed antigen (e.g., HER extracellulardomain) in a biological fluid such as serum [see, e.g., U.S. Pat. No.4,933,294 issued Jun. 12, 1990; WO91/05264 published Apr. 18, 1991; U.S.Pat. No. 5,401,638 issued Mar. 28, 1995; and Sias et al., J. Immunol.Methods 1990; 132: 73-80]. Aside from the above assays, various in vivoassays are available to the skilled practitioner. For example, one mayexpose cells within the body of the patient to an antibody which isoptionally labeled with a detectable label, e.g. a radioactive isotope,and binding of the antibody to cells in the patient can be evaluated,e.g. by external scanning for radioactivity or by analyzing a biopsytaken from a patient previously exposed to the antibody.

Conversely, a cancer which “does not overexpress HER2 receptor” is onewhich does not express higher than normal levels of HER2 receptorcompared to a noncancerous cell of the same tissue type.

A cancer which “overexpresses” a HER ligand is one which producessignificantly higher levels of that ligand compared to a noncancerouscell of the same tissue type. Such overexpression may be caused by geneamplification or by increased transcription or translation.Overexpression of the HER ligand may be determined diagnostically byevaluating levels of the ligand (or nucleic acid encoding it) in thepatient, e.g. in a tumor biopsy or by various diagnostic assays such asthe IHC, FISH, southern blotting, PCR or in vivo assays well known inthe art.

It is contemplated that the HER2 antibody SC formulation in accordancewith this invention may also be used to treat various non-malignantdiseases or disorders, such a include autoimmune disease (e.g.psoriasis); endometriosis; scleroderma; restenosis; polyps such as colonpolyps, nasal polyps or gastrointestinal polyps; fibroadenoma;respiratory disease; cholecystitis; neurofibromatosis; polycystic kidneydisease; inflammatory diseases; skin disorders including psoriasis anddermatitis; vascular disease; conditions involving abnormalproliferation of vascular epithelial cells; gastrointestinal ulcers;Menetrier's disease, secreting adenomas or protein loss syndrome; renaldisorders; angiogenic disorders; ocular disease such as age relatedmacular degeneration, presumed ocular histoplasmosis syndrome, retinalneovascularization from proliferative diabetic retinopathy, retinalvascularization, diabetic retinopathy, or age related maculardegeneration; bone associated pathologies such as osteoarthritis,rickets and osteoporosis; damage following a cerebral ischemic event;fibrotic or edemia diseases such as hepatic cirrhosis, lung fibrosis,carcoidosis, throiditis, hyperviscosity syndrome systemic, OsierWeber-Rendu disease, chronic occlusive pulmonary disease, or edemafollowing burns, trauma, radiation, stroke, hypoxia or ischemia;hypersensitivity reaction of the skin; diabetic retinopathy and diabeticnephropathy; Guillain-Barre syndrome; graft versus host disease ortransplant rejection; Paget's disease; bone or joint inflammation;photoaging (e.g. caused by UV radiation of human skin); benign prostatichypertrophy; certain microbial infections including microbial pathogensselected from adenovirus, hantaviruses, Borrelia burgdorferi, Yersiniaspp. and Bordetella pertussis; thrombus caused by platelet aggregation;reproductive conditions such as endometriosis, ovarian hyperstimulationsyndrome, preeclampsia, dysfunctional uterine bleeding, ormenometrorrhagia; synovitis; atheroma; acute and chronic nephropathies(including proliferative glomerulonephritis and diabetes-induced renaldisease); eczema; hypertrophic scar formation; endotoxic shock andfungal infection; familial adenomatosis polyposis; neurodedenerativediseases (e.g. Alzheimer's disease, AIDS-related dementia, Parkinson'sdisease, amyotrophic lateral sclerosis, retinitis pigmentosa, spinalmuscular atrophy and cerebellar degeneration); myelodysplasticsyndromes; aplastic anemia; ischemic injury; fibrosis of the lung,kidney or liver; T-cell mediated hypersensitivity disease; infantilehypertrophic pyloric stenosis; urinary obstructive syndrome; psoriaticarthritis; and Hasimoto's thyroiditis. Exemplary non-malignantindications for therapy herein include psoriasis, endometriosis,scleroderma, vascular disease (e.g. restenosis, artherosclerosis,coronary artery disease, or hypertension), colon polyps, fibroadenoma orrespiratory disease (e.g. asthma, chronic bronchitis, bronchieactasis orcystic fibrosis).

Where the indication is cancer, the patient may be treated with acombination of the antibody formulation, and a chemotherapeutic agent.The combined administration includes co-administration or concurrentadministration, using separate formulations or a single pharmaceuticalformulation, and consecutive administration in either order, whereinpreferably there is a time period while both (or all) active agentssimultaneously exert their biological activities. Thus, thechemotherapeutic agent may be administered prior to, or following,administration of the antibody formulation in accordance with thepresent invention. In this embodiment, the timing between at least oneadministration of the chemotherapeutic agent and at least oneadministration of the antibody formulation in accordance with thepresent invention is preferably approximately 1 month or less, and mostpreferably approximately 2 weeks or less. Alternatively, thechemotherapeutic agent and the antibody formulation in accordance withthe present invention are administered concurrently to the patient, in asingle formulation or separate formulations.

Treatment with the said antibody formulation will result in animprovement in the signs or symptoms of cancer or disease. For instance,where the disease being treated is cancer, such therapy may result in animprovement in survival (overall survival and/or progression freesurvival) and/or may result in an objective clinical response (partialor complete). Moreover, treatment with the combination of thechemotherapeutic agent and the antibody formulation may result in asynergistic or greater than additive, therapeutic benefit to thepatient.

Normally the antibody in the formulation administered is a nakedantibody. However, the antibody administered may be conjugated with acytotoxic agent. The immunoconjugate and/or antigen to which it is boundis/are then internalized by the cell, resulting in increased therapeuticefficacy of the immunoconjugate in killing the cancer cell to which itbinds. In one embodiment, the cytotoxic agent targets or interferes withnucleic acid in the cancer cell. Examples of such cytotoxic agentsinclude maytansinoids, calioheamicins, ribonucleases and DNAendonucleases. The clinically most advanced immunoconjugates areTrastuzumab-maytansinoid immunoconjugates (T-DM1) as they are describedin WO 2003/037992, in particular the immunoconjugate T-MCC-DM1, thechemical name of which isN^(2′)-deacetyl-N^(2′)-(3-mercapto-1-oxopropyl)-maytansine-4-maleimidomethyl-cyclohexyl-1-carboxyl-Trastuzumab.

For subcutaneous delivery, the formulation may be administered via asuitable device, such as (but not limited to) a syringe; an injectiondevice (e.g. the INJECT-EASE™ and GENJECT™ device); an infusion pump(such as e.g. Accu-Chek™); an injector pen (such as the GENPEN™; anneedleless device (e.g. MEDDECTOR™ and BIOJECTOR™); or via asubcutaneous patch delivery system. A suitable delivery system for theformulations in accordance with the present invention is described in WO2010/029054. Such device comprises about 5 to about 15 ml or moreparticularly 5 ml of the liquid formulation in accordance with thepresent invention.

For the prevention or treatment of disease, the appropriate dosage ofthe antibody will depend on the type of disease to be treated, asdefined above, the severity and course of the disease, whether theantibody is administered for preventive or therapeutic purposes, on theprevious therapy, the patient's clinical history and his response to theantibody, and the discretion of the attending physician. The antibody issuitably administered to the patient at one time or over a series oftreatments. Depending on the type and severity of the disease, about 1μg/kg to 50 mg/kg of bodyweight or more specifically between about 0.1mg/kg to 20 mg/kg of bodyweight) of the anti-HER2 antibody is acandidate initial dosage for administration to the patient, whether, forexample, by one or more separate administrations, or by continuousinfusion. More specifically the dosage of the antibody will be in therange from about 0.05 mg anti-HER2 antibody/kg of bodyweight to about 10mg anti-HER2 antibody/kg of bodyweight. If a chemotherapeutic agent isadministered, it is usually administered at dosages known therefore, oroptionally lowered due to combined action of the drugs or negative sideeffects attributable to administration of the chemotherapeutic agent.Preparation and dosing schedules for such chemotherapeutic agents may beused according to manufacturers' instructions or as determinedempirically by the skilled practitioner. Preparation and dosingschedules for such chemotherapy are also described in ChemotherapyService Ed., M. C. Perry, Williams & Wilkins, Baltimore, Md. (1992).

Other therapeutic regimens may be combined with the antibody including,but not limited to a second (third, fourth, etc) chemotherapeuticagent(s) (in another word a “cocktail” of different chemotherapeuticagents); another monoclonal antibody; a growth inhibitory agent; acytotoxic agent; a chemotherapeutic agent; a EGFR-targeted drug; atyrosine kinase inhibitor; an anti-angiogenic agent; and/or cytokine,etc.; or any suitable combination thereof.

In addition to the above therapeutic regimes, the patient may besubjected to surgical removal of cancer cells and/or radiation therapy.

In another embodiment of the invention, an article of manufacture isprovided which contains the pharmaceutical formulation of the presentinvention and provides instructions for its use. This article ofmanufacture comprises a container. Suitable containers include, forexample, bottles, vials (e.g. multiple or dual chamber vials), syringes(such as multiple or dual chamber syringes) and test tubes. Thecontainer may be formed from a variety of materials such as glass orplastic. The container holds the formulation and the label on, orassociated with, the container may indicate directions for use. Thecontainer holding the formulation may be a multi-use vial, which allowsfor repeat administrations (e.g. from 2 to 6 administrations) of thereconstituted formulation. The article of manufacture may furtherinclude other materials desirable from a commercial and user standpoint,including other buffers, diluents, filters, needles, syringes, andpackage inserts with instructions for use.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of the activeingredient to be effective, and which contains no additional componentswhich are unacceptably toxic to a subject to which the formulation wouldbe administered. Such formulations are sterile.

A “sterile” formulation is aseptic or free from all livingmicroorganisms and their spores.

A “stable” formulation is one in which all the protein thereinessentially retain their physical stability and/or chemical stabilityand/or biological activity upon storage at the intended storagetemperature, e.g. 2-8° C. It is desired that the formulation essentiallyretains its physical and chemical stability, as well as its biologicalactivity upon storage. The storage period is generally selected based onthe intended shelf-life of the formulation. Furthermore, the formulationshould be stable following freezing (to, e.g., −70° C.) and thawing ofthe formulation, for example following 1, 2 or 3 cycles of freezing andthawing. Various analytical techniques for measuring protein stabilityare available in the art and are reviewed in Peptide and Protein DrugDelivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y.,Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993), forexample. Stability can be measured at a selected temperature for aselected time period. Stability can be evaluated qualitatively and/orquantitatively in a variety of different ways, including evaluation ofaggregate formation (for example using size exclusion chromatography, bymeasuring turbidity, and/or by visual inspection); by assessing chargeheterogeneity using cation exchange chromatography or capillary zoneelectrophoresis; amino-terminal or carboxy-terminal sequence analysis;mass spectrometric analysis; SDS-PAGE analysis to compare reduced andintact antibody; peptide map (for example tryptic or LYS-C) analysis;evaluating biological activity or antigen binding function of theantibody; etc. Instability may involve any one or more of: aggregation,deamidation (e.g. Asn deamidation), oxidation (e.g. Met oxidation),isomerization (e.g. Asp isomeriation), clipping/hydrolysis/fragmentation(e.g. hinge region fragmentation), succinimide formation, unpairedcysteine(s), N-terminal extension, C-terminal processing, glycosylationchanges, etc. A “deamidated” monoclonal antibody herein is one in whichone or more asparagine residue thereof has been modified, e.g. to anaspartic acid or an iso-aspartic acid by a post-translationalmodification.

As used herein the term “buffering agent providing a pH of 5.5±2.0”refers to an agent which provides that the solution comprising itresists changes in pH by the action of its acid/base conjugatecomponents. The buffer used in the formulations in accordance with thepresent invention has a pH in the range from about 5.0 to about 7.0, orfrom about 5.0 to about 6.5, or from about 5.3 to about 5.8. A pH ofabout 5.5 has to be found to be most suitable. Examples of bufferingagents that will control the pH in this range include acetate,succinate, gluconate, histidine, citrate, glycylglycine and otherorganic acid buffers. The most suitable buffer in accordance with thepresent invention is a histidine buffer, such as e.g. L-histidine/HCl.

A “histidine buffer” is a buffer comprising the amino acid histidine.Examples of histidine buffers include histidine chloride, histidineacetate, histidine phosphate, histidine sulfate. The histidine bufferidentified in the examples as being most suitable is a histidinechloride buffer. Such histidine chloride buffer is prepared by titratingL-histidine (free base, solid) with diluted hydrochloric acid. Inparticular the histidine buffer or histidine chloride buffer is at pH of5.5±0.6, more particularly at a pH from about 5.3 to about 5.8, and mostparticularly has a pH of 5.5.

By “isotonic” is meant that the formulation of interest has essentiallythe same osmotic pressure as human blood. Isotonic formulations willgenerally have an osmotic pressure from about 250 to 350 mOsm.Isotonicity can be measured using a vapor pressure or freezing-pointdepression type osmometer.

A “saccharide” herein comprises the general composition (CH₂O)_(n) andderivatives thereof, including monosaccharides, disaccharides,trisaccharides, polysaccharides, sugar alcohols, reducing sugars,nonreducing sugars, etc. Examples of saccharides herein include glucose,sucrose, trehalose, lactose, fructose, maltose, dextran, glycerin,dextran, erythritol, glycerol, arabitol, sylitol, sorbitol, mannitol,mellibiose, melezitose, raffinose, mannotriose, stachyose, maltose,lactulose, maltulose, glucitol, maltitol, lactitol, iso-maltulose, etc.Particularly the formulations described herein comprise a non-reducingdisaccharide as a stabilizing agent, such as a saccharide selected fromthe group of trehalose (e.g. in the form of α,α-trehalose dihydrate) andsucrose.

Herein, a “surfactant” refers to a surface-active agent, e.g. a nonionicsurfactant. Examples of surfactants herein include polysorbate (forexample, polysorbate 20 and, polysorbate 80); poloxamer (e.g. poloxamer188); Triton; sodium dodecyl sulfate (SDS); sodium laurel sulfate;sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, orstearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- orstearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine;lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-,myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g.lauroamidopropyl); myristamidopropyl-, palmidopropyl-, orisostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodiummethyl oleyl-taurate; and the MONAQU AT™ series (Mona Industries, Inc.,Paterson, N.J.); polyethyl glycol, polypropyl glycol, and copolymers ofethylene and propylene glycol (e.g. Pluronics, PF68 etc); etc.Polysorbate 20 (PS20) and Polysorbate 80 (PS80), respectively have beenfound to be particularly suitable in the formulations described herein.

The term “antibody” herein is used in the broadest sense andspecifically covers full length monoclonal antibodies, polyclonalantibodies, multispecific antibodies (e.g. bispecific antibodies) formedfrom at least two full length antibodies, and antibody fragments, solong as they exhibit the desired biological activity.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variants that mayarise during production of the monoclonal antibody, such variantsgenerally being present in minor amounts. In contrast to polyclonalantibody preparations that typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody is directed against a single determinant on the antigen. Inaddition to their specificity, the monoclonal antibodies areadvantageous in that they are uncontaminated by other immunoglobulins.The modifier “monoclonal” indicates the character of the antibody asbeing obtained from a substantially homogeneous population ofantibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method first described by Köhler et al, Nature,256:495 (1975), or may be made by recombinant DNA methods (see, e.g.,U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also beisolated from phage antibody libraries using the techniques described inClarkson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol.Biol., 222:581-597 (1991).

An “antibody fragment” comprises a portion of a full length antibody, inparticular comprises the antigen-binding or variable region thereof.Examples of antibody fragments include Fab, Fab′, F(ab′)₂, and Fvfragments; diabodies; linear antibodies; single-chain antibodymolecules; and multispecific antibodies formed from antibodyfragment(s).

A “full length antibody” is one which comprises an antigen-bindingvariable region as well as a light chain constant domain (CL) and heavychain constant domains, CH1, CH2 and CH3. The constant domains may benative sequence constant domains (e.g. human native sequence constantdomains) or amino acid sequence variants thereof. In particular the fulllength antibody has one or more effector functions.

An “amino acid sequence variant” antibody herein is an antibody with anamino acid sequence which differs from a main species antibody.Ordinarily, amino acid sequence variants will possess at least about 70%homology with the main species antibody, and preferably, they will be atleast about 80%, more preferably at least about 90% homologous with themain species antibody. The amino acid sequence variants possesssubstitutions, deletions, and/or additions at certain positions withinor adjacent to the amino acid sequence of the main species antibody.Examples of amino acid sequence variants herein include acidic variant(e.g. deamidated antibody variant), basic variant, the antibody with anamino-terminal leader extension (e.g. VHS-) on one or two light chainsthereof, antibody with a C-terminal lysine residue on one or two heavychains thereof, etc, and includes combinations of variations to theamino acid sequences of heavy and/or light chains. The antibody variantof particular interest herein is the antibody comprising anamino-terminal leader extension on one or two light chains thereof,optionally further comprising other amino acid sequence and/orglycosylation differences relative to the main species antibody.

A “glycosylation variant” antibody herein is an antibody with one ormore carbohydrate moieties attached thereto which differ from one ormore carbohydrate moieties attached to a main species antibody. Examplesof glycosylation variants herein include antibody with a G1 or G2oligosaccharide structure, instead a G0 oligosaccharide structure,attached to an Fc region thereof, antibody with one or two carbohydratemoieties attached to one or two light chains thereof, antibody with nocarbohydrate attached to one or two heavy chains of the antibody, etc,and combinations of glycosylation alterations. Moreover the term“glycosylation variant” includes also glycoengineered antibodies such asthose described in EP 1'331'266 and U.S. Pat. No. 7,517,670.

Antibody “effector functions” refer to those biological activitiesattributable to the Fc region (a native sequence Fc region or amino acidsequence variant Fc region) of an antibody. Examples of antibodyeffector functions include C1q binding; complement dependentcytotoxicity (CDC); Fc receptor binding; antibody-dependentcell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cellsurface receptors (e.g. B cell receptor; BCR), etc.

Depending on the amino acid sequence of the constant domain of theirheavy chains, full length antibodies can be assigned to different“classes”. There are five major classes of full length antibodies: IgA,IgD, IgE, IgG, and IgM, and several of these may be further divided into“subclasses” (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.The heavy-chain constant domains that correspond to the differentclasses of antibodies are called α [alpha], δ [delta], ε [epsilon], γ[gamma], and μ [mu], respectively. The subunit structures andthree-dimensional configurations of different classes of immunoglobulinsare well known.

Herein, “biological activity” of a monoclonal antibody refers to theability of the antibody to bind to antigen and result in a measurablebiological response which can be measured in vitro or in vivo. Suchactivity may be antagonistic (for example where the antibody is a HER2antibody) or agonistic. In the case of Pertuzumab, in one embodiment,the biological activity refers to the ability of the formulated antibodyto inhibit proliferation of the human breast cancer cell lineMDA-MB-175-VII.

The term “monoclonal antibodies” herein specifically include theso-called chimeric antibodies in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567;and Morrison et al, Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).Chimeric antibodies of interest herein include “primatized” antibodiescomprising variable domain antigen-binding sequences derived from anon-human primate (e.g. Old World Monkey, Ape etc) and human constantregion sequences.

“Humanized” forms of non-human (e.g., rodent) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992).

Humanized HER2 antibodies include huMAb4D5-1, huMAb4D5-2, huMAb4D5-3,huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8 orTrastuzumab (HERCEPTIN™) as described in Table 3 of U.S. Pat. No.5,821,337; humanized 520C9 (WO93/21319) and humanized 2C4 antibodies,such as Pertuzumab as described further herein below.

For the purposes herein, “Trastuzumab”, “HERCEPTIN™” and “huMAb4D5-8”refer to an anti-HER2 antibody directed against the 4D5 epitope. Suchantibody preferably comprises the light and heavy chain amino acidsequences disclosed e.g. in FIG. 14 of WO 2006/044908.

The “epitope 4D5” is the region in the extracellular domain of HER2 towhich the antibody 4D5 (ATCC CRL 10463) and Trastuzumab bind. Thisepitope is close to the transmembrane domain of HER2, and within DomainIV of HER2. To screen for antibodies which bind to the 4D5 epitope, aroutine cross-blocking assay such as that described in Antibodies, ALaboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and DavidLane (1988), can be performed. Alternatively, epitope mapping can beperformed to assess whether the antibody binds to the 4D5 epitope ofHER2 (e.g. any one or more residues in the region from about residue 529to about residue 625, inclusive, of HER2). The “epitope 7C2/7F3” is theregion at the amino terminus, within Domain I, of the extracellulardomain of HER2 to which the 7C2 and/or 7F3 antibodies bind. To screenfor antibodies which bind to the 7C2/7F3 epitope, a routinecross-blocking assay such as that described in “Antibodies, A LaboratoryManual” (Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988))can be performed. Alternatively, epitope mapping can be performed toestablish whether the antibody binds to the 7C2/7F3 epitope on HER2(e.g. any one or more of residues in the region from about residue 22 toabout residue 53 of HER2).

Herein, “Pertuzumab” and “rhuMAb 2C4” refer to an antibody that binds tothe 2C4 epitope and preferably comprising the variable light andvariable heavy amino acid sequences disclosed in WO 2006/044908, moreparticularly the humanized 2C4 version 574 disclosed in FIG. 2 of WO2006/044908.

The “epitope 2C4” is the region in the extracellular domain of HER2 towhich the antibody 2C4 binds. In order to screen for antibodies whichbind to the 2C4 epitope, a routine cross-blocking assay such as thatdescribed in Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory, Ed Harlow and David Lane (1988), can be performed.Alternatively, epitope mapping can be performed to assess whether theantibody binds to the 2C4 epitope of HER2. Epitope 2C4 comprisesresidues from domain II in the extracellular domain of HER2. 2C4 andPertuzumab bind to the extracellular domain of HER2 at the junction ofdomains I, II and III (Franklin et al. Cancer Cell 5:317-328 (2004)).

A “growth inhibitory agent” when used herein refers to a compound orcomposition which inhibits growth of a cell, especially a HER expressingcancer cell either in vitro or in vivo. Thus, the growth inhibitoryagent may be one which significantly reduces the percentage of HERexpressing cells in S phase. Examples of growth inhibitory agentsinclude agents that block cell cycle progression (at a place other thanS phase), such as agents that induce G1 arrest and M-phase arrest.Classical M-phase blockers include the vincas (vincristine andvinblastine), taxanes, and topo II inhibitors such as doxorubicin,epirubicin, daunorubicin, etoposide, and bleomycin. Those agents thatarrest G1 also spill over into S-phase arrest, for example, DNAalkylating agents such as tamoxifen, prednisone, dacarbazine,mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C.Further information can be found in “The Molecular Basis of Cancer”,Mendelsohn and Israel, eds., Chapter 1, entitled “Cell cycle regulation,oncogenes, and antineoplastic drugs” by Murakami et al. (WB Saunders:Philadelphia, 1995), especially p. 13.

Examples of “growth inhibitory” antibodies are those which bind to HER2and inhibit the growth of cancer cells overexpressing HER2. Preferredgrowth inhibitory HER2 antibodies inhibit growth of SK-BR-3 breast tumorcells in cell culture by greater than 20%, and preferably greater than50% (e.g. from about 50% to about 100%) at an antibody concentration ofabout 0.5 to 30 μg/ml, where the growth inhibition is determined sixdays after exposure of the SK-BR-3 cells to the antibody (see U.S. Pat.No. 5,677,171 issued Oct. 14, 1997). The preferred growth inhibitoryantibody is a humanized variant of murine monoclonal antibody 4D5, e.g.,Trastuzumab.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures. Those in need of treatment include those alreadywith the disease as well as those in which the disease is to beprevented. Hence, the patient to be treated herein may have beendiagnosed as having the disease or may be predisposed or susceptible tothe disease.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g. At²¹¹,I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³² and radioactiveisotopes of Lu), chemotherapeutic agents, and toxins such as smallmolecule toxins or enzymatically active toxins of bacterial, fungal,plant or animal origin, including fragments and/or variants thereof.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN™);alkyl sulfonates such as busulfan, improsulfan and piposulfan;aziridines such as benzodopa, carboquone, meturedopa, and uredopa;ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide and trimethylolomelarnine; acetogenins(especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol(dronabinol, MARENOL™); beta-lapachone; lapachol; colchicines; betulinicacid; a camptothecin (including the synthetic analogue topotecan(HYCAMTIN™), CPT-11 (irinotecan, C AMPTOSAR™), acetylcamptothecin,scopolectin, and 9-aminocamptothecin); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); podophyllotoxin; podophyllinic acid; teniposide;cryptoplhycins (particularly cryptophycin 1 and cryptophycin 8);dolastatin; duocarmycin (including the synthetic analogues, KW-2189 andCB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin;nitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfa[pi]nide, uracil mustard;nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine,nimustine, and ranirnnustine; antibiotics such as the enediyneantibiotics (e. g., calicheamicin, especially calicheamicin gamma 11 andcalicheamicin omega 11 (see, e.g., Angew, Chemie Intl. Ed. Engl., 33:183-186 (1994)); dynemicin, including dynemicin A; an esperamicin; aswell as neocarzinostatin chromophore and related chromoprotein enediyneantibiotic chromophores), aclacinomysins, actinomycin, authramycin,azaserine, bleomycins, cactinornycin, carabicin, carminomycin,carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin (including ADRIAMYCIN™,morpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin, doxorubicin HCl liposome injection (DOXIL™),liposomal doxorubicin TLC D-99 (MYOCET™), peglylated liposomaldoxorubicin (CAELYX™), and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolicacid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate,gemcitabine (GEMZAR™), tegafur (UFTORAL™), capecitabine (XELODA™), anepothilone, and 5-fluorouracil (5-FU); folic acid analogues such asdenopterin, methotrexate, pteropterin, trimetrexate; purine analogs suchas fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;anti-adrenals such as aminoglutethimide, mitotane, trilostane; folicacid replenisher such as frolinic acid; aceglatone; aldophosphamideglycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil;bisantrene; edatraxate; defofamine; demecolcine; diaziquone;elfornithine; elliptinium acetate; etoglucid; gallium nitrate;hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine andansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine;pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide;procarbazine; PSKL™ polysaccharide complex (JHS Natural Products,Eugene, Oreg.); razoxane; rhizoxin; sizofiran; spirogermaraium;tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine;trichothecenes (especially T-2 toxin, verracurin A, roridin A andanguidine); urethan; dacarbazine; mannomustine; mitobronitol;mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); thiotepa;taxoid, e.g., paclitaxel (TAXOL™), albumin-engineered nanoparticleformulation of paclitaxel (ABRAXANE™), and docetaxel (TAXOTERE™);chloranbucil; 6-thioguanine; mercaptopurine; metliotrexate; platinumagents such as cisplatin, oxaliplatin, and carboplatin; vincas, whichprevent tubulin polymerization from forming microtubules, includingvinblastine (VELBAN™), vincristine (ONCOVIN™), vindesine (ELDISINE™),FILDESIN™), and vinorelbine (NAVELBINE™)); etoposide (VP-16);ifosfamide; mitoxantrone; leucovovin; novantrone; edatrexate;daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000;difluorometlhylornithine (DMFO); retinoids such as retinoic acid,including bexarotene (TARGRETIN™); bisphosphonates such as clodronate(for example, BONEFOS™ or OSTAC™), etidronate (DIDROCAL™), NE-58095,zoledronic acid/zoledronate (ZOMETA™), alendronate (FOSAMAJX™),pamidronate (AREDIA™), tiludronate (SKELID™), or risedronate (ACTONEL™);troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisenseoligonucleotides, particularly those that inhibit expression of genes insignaling pathways implicated in aberrant cell proliferation, such as,for example, PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor(EGF-R); vaccines such as THERATOPE™ vaccine and gene therapy vaccines,for example, ALLOVECTIN™ vaccine, LEUVECTIN™ vaccine, and VAXID™vaccine; topoisomerase 1 inhibitor (e.g., LURTOTECAN™); rmRH (e.g.,ABARELIX™); BAY439006 (sorafenib; Bayer); SU-11248 (Pfizer); perifosine,COX-2 inhibitor (e.g. celecoxib or etoricoxib), proteosome inhibitor(e.g. PS341); bortezomib (VELCADE™); CCI-779; tipifarnib (R1 1577);orafenib, ABT510; Bcl-2 inhibitor such as oblimersen sodium(GENASENSE™); pixantrone; EGFR inhibitors (see definition below);tyrosine kinase inhibitors (see definition below); and pharmaceuticallyacceptable salts, acids or derivatives of any of the above; as well ascombinations of two or more of the above such as CHOP, an abbreviationfor a combined therapy of cyclophosphamide, doxorubicin, vincristine,and prednisolone, and FOLFOX, an abbreviation for a treatment regimenwith oxaliplatin (ELOXATIN™) combined with 5-FU and leucovovin.

Also included in this definition are anti-hormonal agents that act toregulate or inhibit hormone action on tumors such as anti-estrogens withmixed agonist/antagonist profile, including, tamoxifen (NOLVADEX™),4-hydroxytamoxifen, toremifene (FARESTON™), idoxifene, droloxifene,raloxifene (EVTSTA™), trioxifene, keoxifene, and selective estrogenreceptor modulators (SERMs) such as SERM3; pure anti-estrogens withoutagonist properties, such as fulvestrant (FASLODEX™), and EM800 (suchagents may block estrogen receptor (ER) dimerization, inhibit DNAbinding, increase ER turnover, and/or suppress ER levels); aromataseinhibitors, including steroidal aromatase inhibitors such as formestaneand exemestane (AROMASIN™), and nonsteroidal aromatase inhibitors suchas anastrazole (ARIMIDEX™), letrozole (FEMARA™) and aminoglutethimide,and other aromatase inhibitors including vorozole (RIVISOR™), megestrolacetate (MEGASE™), fadrozole, imidazole; lutenizing hormone-releasinghormone agonists, including leuprolide (LUPRON™ and ELIGARD™),goserelin, buserelin, and tripterelin; sex steroids, includingprogestines such as megestrol acetate and medroxyprogesterone acetate,estrogens such as diethylstilbestrol and premarin, andandrogens/retinoids such as fluoxymesterone, all transretionic acid andfenretinide; onapristone; anti-progesterones; estrogen receptordown-regulators (ERDs); anti-androgens such as flutamide, nilutamide andbicalutamide; testolactone; and pharmaceutically acceptable salts, acidsor derivatives of any of the above; as well as combinations of two ormore of the above.

As used herein, the term “EGFR-targeted drug” refers to a therapeuticagent that binds to EGFR and, optionally, inhibits EGFR activation.Examples of such agents include antibodies and small molecules that bindto EGFR. Examples of antibodies which bind to EGFR include MAb 579 (ATCCCRL HB 8506), MAb 455 (ATCC CRL HB8507), MAb 225 (ATCC CRL 8508), MAb528 (ATCC CRL 8509) (see, U.S. Pat. No. 4,943,533, Mendelsohn et al.)and variants thereof, such as chimerized 225 (C225 or Cetuximab;ERBITUX™) and reshaped human 225 (H225) (see, WO 96/40210, ImcloneSystems Inc.); antibodies that bind type II mutant EGFR (U.S. Pat. No.5,212,290); humanized and chimeric antibodies that bind EGFR asdescribed in U.S. Pat. No. 5,891,996; and human antibodies that bindEGFR, such as ABX-EGF (see WO98/50433, Abgenix). The anti-EGFR antibodymay be conjugated with a cytotoxic agent, thus generating animmunoconjugate (see, e.g., EP-A-659439, Merck patent GmbH). Examples ofsmall molecules that bind to EGFR include ZD 1839 or Gefitinib (IRESSA™;Astra Zeneca), CP-358774 or Erlotinib HCl (TARCEVA™ Genentech/Roche/OSI)and AG1478, AG1571 (SU 5271; Sugen)

A “tyrosine kinase inhibitor” is a molecule which inhibits to someextent tyrosine kinase activity of a tyrosine kinase such as a HERreceptor. Examples of such inhibitors include the EGFR-targeted drugsnoted in the preceding paragraph as well as small molecule HER2 tyrosinekinase inhibitor such as TAK1 65 available from Takeda, dual-HERinhibitors such as EKB-569 (available from Wyeth) which preferentiallybinds EGFR but inhibits both HER2 and EGFR-overexpressing cells,GW572016 (available from Glaxo) an oral HER2 and EGFR tyrosine kinaseinhibitor, and PKI-166 (available from Novartis); pan-HER inhibitorssuch as canertinib (CI-1033; Pharmacia); Raf-1 inhibitors such asantisense agent ISIS-5132 available from ISIS Pharmaceuticals whichinhibits Raf-1 signaling; non-HER targeted TK inhibitors such asImatinib mesylate (Gleevec™) available from Novartis; MAPK extracellularregulated kinase I inhibitor CI-1040 (available from Pharmacia);quinazolines, such as PD 153035,4-(3-chloroanilino) quinazoline;pyridopyrimidines; pyrimidopyrimidines; pyrrolopyrimidines, such as CGP59326, CGP 60261 and CGP 62706; pyrazolopyrimidines,4-(phenylamino-7H-pyrrolo[2,3-d] pyrimidines; curcumin (diferuloylmethane, 4,5-bis (4-fluoroanilino)phthalimide); tyrphostines containingnitrothiophene moieties; PD-0183805 (Warner-Larnber); antisensemolecules (e.g. those that bind to HER-encoding nucleic acid);quinoxalines (U.S. Pat. No. 5,804,396); tryphostins (U.S. Pat. No.5,804,396); ZD6474 (Astra Zeneca); PTK-787 (Novartis/Schering AG);pan-HER inhibitors such as CI-1033 (Pfizer); Affinitac (ISIS 3521;Isis/Lilly); PKI 166 (Novartis); GW2016 (Glaxo SmithKline); CI-1033(Pfizer); EKB-569 (Wyeth); Semaxinib (Sugen); ZD6474 (AstraZeneca);PTK-787 (Novartis/Schering AG); INC-IC11 (Imclone); or as described inany of the following patent publications: U.S. Pat. No. 5,804,396;WO99/09016 (American Cyanamid); WO98/43960 (American Cyanamid);WO97/38983 (Warner Lambert); WO99/06378 (Warner Lambert); WO99/06396(Warner Lambert); WO96/30347 (Pfizer, Inc); WO96/33978 (Zeneca);WO96/3397 (Zeneca); and WO96/33980 (Zeneca).

An “anti-angiogenic agent” refers to a compound which blocks, orinterferes with to some degree, the development of blood vessels. Theanti-angiogenic factor may, for instance, be a small molecule orantibody that binds to a growth factor or growth factor receptorinvolved in promoting angiogenesis. The preferred anti-angiogenic factorherein is an antibody that binds to Vascular Endothelial Growth Factor(VEGF), such as Bevacizumab (AVASTIN™).

The term “cytokine” is a generic term for proteins released by one cellpopulation which act on another cell as intercellular mediators.Examples of such cytokines are lymphokines, monokines, and traditionalpolypeptide hormones. Included among the cytokines are growth hormonesuch as human growth hormone, N-methionyl human growth hormone, andbovine growth hormone, parathyroid hormone, thyroxine, insulin,proinsulin, relaxin; prorelaxin, glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH), hepatic growth factor; fibroblast growthfactor, prolactin, placental lactogen, tumor necrosis factor α and β,mullerian-inhibiting substance, mouse gonadotropin-associated peptide,inhibin; activin, vascular endothelial growth factor, integrin,thrombopoietin (TPO), nerve growth factors such as NGF-β,platelet-growth factor; transforming growth factors (TGFs) such as TGF-αand TGF-β, insulin-like growth factor-I and -II, erythropoietin (EPO),osteoinductive factors; interferons such as interferon-α, -β, and -γ,colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF),granulocyte-macrophage-CSF (GM-CSF), and granulocyte-CSF (G-CSF),interleukins (ILs) such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, a tumor necrosis factor such asTNF-α or TNF-β, and other polypeptide factors including LIF and kitligand (KL). As used herein, the term cytokine includes proteins fromnatural sources or from recombinant cell culture and biologically activeequivalents of the native sequence cytokines.

The term “effective amount” refers to an amount which provides thedesired effect. In the case of a formulation ingredient such as thehyaluronidase enzyme in accordance with the present invention aneffective amount is the amount necessary to increase the dispersion andabsorption of the co-administered anti-HER2 antibody in such a way thatthe anti-HER2 antibody can act in a therapeutically effective way asoutlined above. In the case of a pharmaceutical drug substance it is theamount of active ingredient effective to treat a disease in the patient.Where the disease is cancer, the effective amount of the drug may reducethe number of cancer cells; reduce the tumor size; inhibit (i.e., slowto some extent and preferably stop) cancer cell infiltration intoperipheral organs; inhibit (i.e., slow to some extent and preferablystop) tumor metastasis; inhibit, to some extent, tumor growth; and/orrelieve to some extent one or more of the symptoms associated with thecancer. To the extent the drug may prevent growth and/or kill existingcancer cells, it may be cytostatic and/or cytotoxic. The effectiveamount may extend progression free survival, result in an objectiveresponse (including a partial response, PR, or complete response, CR),increase overall survival time, and/or improve one or more symptoms ofcancer.

The antibody which is formulated in accordance with the presentinvention is preferably essentially pure and desirably essentiallyhomogeneous (i.e. free from contaminating proteins etc, whereby thehyaluronidase enzyme in the formulation in accordance of this inventionis not to be considered to be a contaminating protein of the anti-HER2monoclonal antibody in accordance of the present invention). An“essentially pure” antibody means a composition comprising at leastabout 90% by weight of the antibody, based on total weight of thecomposition, preferably at least about 95% by weight. An “essentiallyhomogeneous” antibody means a composition comprising at least about 99%by weight of antibody, based on total weight of the composition.

The invention will be more fully understood by reference to thefollowing Examples. They should not, however, be construed as limitingthe scope of the invention. All literature and patent citations areincorporated herein by reference.

The Examples are further illustrated by the appended FIGS. 1-4.

EXAMPLES

The anti-HER2 formulations for subcutaneous administration according tothe invention were developed based on the experimental results asprovided below using the general preparatory and analytical methods andassays as outlined below.

A) Preparation of the Components for the Formulation

Trastuzumab is manufactured by techniques generally known for theproduction of recombinant proteins. A genetically engineered Chinesehamster ovary (CHO) cell line prepared as described in EP-B-590058 isexpanded in cell culture from a master cell bank. The Trastuzumabmonoclonal antibody is harvested from the cell culture fluid andpurified using immobilized Protein A affinity chromatography, cationexchange chromatography (e.g. SP-Sepharose FF), a filtration step toremove viral contaminations (e.g. a PVDF membrane (sold by Miliporeunder the name Viresolve filters), followed by anion exchangechromatography (e.g. Q-Sepharose FF) and anultrafiltration/diafiltration step. For preparing the formulations inaccordance with these examples the Trastuzumab was provided at aconcentration of approx. 100 mg/ml in a 20 mM histidine buffer at a pHof approximately 6.0.

rHuPH20 is manufactured by techniques generally known from theproduction of recombinant proteins. The process begins with thawing ofcells from the working cell bank (WCB) or master cell bank (MCB) andexpansion through cell culture in a series of spinner flasks followed byexpansion in a bioreactor. After completion of the production phase, thecell culture liquid is clarified by filtration, and is then treated withsolvent/detergent to inactivate viruses. The protein is then purified bya series of column chromatography processes to remove process andproduct related impurities. A viral filtration step is performed, andthe filtered bulk is then concentrated, formulated into the finalbuffer: 10 mg/mL rHuPH20 in 20 mM L-histidine/HCl buffer, pH 6.5, 130 mMNaCl, 0.05% (w/v) polysorbate 80. The rHuPH20 bulk is stored below −70°C.

The other excipients of the formulation in accordance with the presentinvention are widely used in the practice and known to the personskilled in the art. There is therefore no need to be explained them herein detail.

Liquid drug product formulations for subcutaneous administrationaccording to the invention were developed as follows.

Example 1: Preparation of the Liquid Formulations

For the preparation of the liquid formulations Trastuzumab wasbuffer-exchanged against a diafiltration buffer containing theanticipated buffer composition and, when required, concentrated bydiafiltration to an antibody concentration of approx. 150 mg/ml. Aftercompletion of the diafiltration operation, the excipients (e.g.trehalose, rHuPH20) were added as stock solutions to the antibodysolution. The surfactant was then added as a 50 to 200-fold stocksolution. Finally the protein concentration was adjusted with a bufferto the final Trastuzumab concentration of about 110 mg/ml, 120 mg/ml or130 mg/ml as specified in the particular formulations further below.

All formulations were sterile-filtered through 0.22 μm low proteinbinding filters and aseptically filled into sterile 6 ml glass vialsclosed with ETFE (Copolymer of ethylene and tetrafluoroethylene)-coatedrubber stoppers and alucrimp caps. The fill volume was approx. 3.0 ml.These formulations were stored at different climate conditions (5° C.,25° C. and 30° C.) for different intervals of time and stressed byshaking (1 week at a shaking frequency of 200 min⁻¹ at 5° C. and 25° C.)and freeze-thaw stress methods. The samples were analyzed before andafter applying the stress tests by the following analytical methods:

1) UV spectrophotometry;

2) Size Exclusion Chromatography (SEC);

3) by Ion exchange chromatography (IEC);4) by turbidity of the solution;5) for visible particles; and6) for rHuPH20 activity.

UV spectroscopy, used for determination of protein content, wasperformed on a Perkin Elmer λ35 UV spectrophotometer in a wavelengthrange from 240 nm to 400 nm. Neat protein samples were diluted toapprox. 0.5 mg/ml with the corresponding formulation buffer. The proteinconcentration was calculated according to Equation 1.

$\begin{matrix}{{{Protein}\mspace{14mu} {content}} = \frac{{A(280)} - {{A(320)} \times {{dil}.\mspace{11mu} {factor}}}}{ɛ{\langle{{cm}^{2}\text{/}{mg}}\rangle} \times d{\langle{cm}\rangle}}} & {{Equation}\mspace{14mu} 1}\end{matrix}$

The UV light absorption at 280 nm was corrected for light scattering at320 nm and multiplied with the dilution factor, which was determinedfrom the weighed masses and densities of the neat sample and thedilution buffer. The numerator was divided by the product of thecuvette's path length d and the extinction coefficient E.

Size Exclusion Chromatography (SEC) was used to detect soluble highmolecular weight species (aggregates) and low molecular weighthydrolysis products (LMW) in the formulations. The method was performedon a Waters Alliance 2695 HPLC instrument with a Waters W2487 DualAbsorbance Detector and equipped with 2 TosoHaas TSK Gel SuperSW3000,4.6×300 mm columns in row. Intact monomer, aggregates and hydrolysisproducts were separated by an isocratic elution profile, using 50 mMsodium phosphate, 420 mM sodium perchlorate, pH 7.0 as mobile phase, andwere detected at a wavelength of 280 nm.

Ion Exchange Chromatography (IEC) was performed to detect chemicaldegradation products altering the net charge of Trastuzumab in theformulations. For this purpose Trastuzumab was digested withCarboxpeptidase B. The method used a suitable HPLC instrument equippedwith a UV detector (detection wavelength 214 nm) and a Dionex ProPac™WCX-10 Analytical cation-exchange column (4×250 mm). 10 mM sodiumphosphate buffer pH 7.5 in H₂O and 10 mM sodium phosphate buffer pH 7.5and 100 mM NaCl were used as mobile phases A and B, respectively, with aflow rate of 0.8 ml/min.

For the determination of the turbidity, opalescence was measured in FTU(turbidity units) using a HACH 2100AN turbidimeter at room temperature.

Samples were analyzed for visible particles by using a Seidenader V90-Tvisual inspection instrument.

An in vitro enzyme assay of rHuPH20 as hyaluronidase was used asactivity assay. The assay is based on the formation of an insolubleprecipitate when hyaluronan (sodium hyaluronate) binds to a cationicprecipitant. Enzyme activity was measured by incubating rHuPH20 withhyaluronan substrate and then precipitating the undigested hyaluronanwith acidified serum albumin (horse serum). The turbidity was measuredat a wavelength of 640 nm and the decrease in turbidity resulting fromenzyme activity on the hyaluronan substrate is a measure of the enzymeactivity. The procedure is run using a standard curve generated withdilutions of rHuPH20 assay reference standard, and sample activity isread from the curve.

Further experiments were performed including following variations:

-   -   variations in pH from about approx. 5.0 to approx. 6.0    -   variations in protein content from about approx. 110 mg/ml to        approx. 130 mg/ml    -   variations in surfactant from approx. 0.02% to approx. 0.06%    -   variations in stabilizer (methionine) from about 5 mM to about        15 mM

The compositions and the results of the stability testing for the liquidanti-HER-2 drug product formulations (Formulations A to X) are providedin Table 1 below wherein the following abbreviations are used:

ffp:=free from particles; effp:=essentially free from particles;wafp:=with a few particles F/T:=freezing/thawing; skg:=shaking; nd:=notdetermined

The formulations specified below show that it is possible to provideliquid formulations with high concentrations of two different proteins.Such formulations can be prepared with greater ease and lower costs thanlyophilized formulations. Moreover such formulations are easier tohandle as no dissolving of the lyophilized final product(reconstitution) is required (ready to use). It has been found that theformulations specified in Tables 1, 3 and 4 are also suitable for theformulation of highly concentrated, stable pharmaceutical formulation ofpharmaceutically active anti-HER2 antibodies lacking the hyaluronidaseenzyme. Therefore, in one aspect the present invention relates also toformulations having the specified ingredients but lacking thehyaluronidase enzyme.

TABLE 1 Composition and stability data of liquid anti-HER2 drug productformulations according to this invention Protein Size Exclusion-HPLC IonExchange-HPLC HE Storage Storage concentration HMW Monomer LMW Main PeakPeak 1 Peak 4 Turbidity Visible activity condition Time (mg/ml) (%) (%)(%) (%) (%) (%) (FTU) particles (U/ml) Formulation A is a liquidformulation with the composition 120 mg/ml Trastuzumab, 20 mML-histidine/HCl pH 5.5, 210 mM α,α-trehalose dihydrate, 10 mMmethionine, 0.04% polysorbate 20, 12′000 U/ml rHuPH20. — Initial 111 0.498.9 0.7 60 15 10 5.0 ffp 13613 Skg 5° C. 1 week nd 0.4 98.7 0.9 Nd ndnd 5.2 ffp 13775 Skg 25° C. 1 week nd 0.4 98.7 0.9 60 11 12 5.7 ffp12053 F/T (5 cycles) nd 0.4 98.7 0.9 Nd nd nd 6.0 effp 12558  5° C. 8weeks 105 0.4 98.5 1.1 60 15 11 4.8 ffp 13204 21 weeks nd 0.4 98.7 0.862 12 11 5.3 ffp 14940 36 weeks nd 0.4 98.7 0.8 59 11 12 3.8 ffp 1261325° C. 8 weeks 106 0.5 98.3 1.2 53 8 19 4.7 ffp 12176 21 weeks nd 0.697.7 1.7 42 7 26 5.2 ffp 13976 36 weeks nd 0.7 97.2 2.1 33 7 34 3.8 ffp12348 30° C. 8 weeks 105 0.6 97.8 1.6 45 6 26 4.4 ffp 13294 21 weeks 1070.8 96.3 2.9 33 6 25 5.5 ffp nd Formulation B is a liquid formulationwith the composition 120 mg/ml Trastuzumab, 20 mM L-histidine/HCl pH5.5, 210 mM α,α-trehalose dihydrate, 10 mM methionine, 0.06% polysorbate80, 12′000 U/ml rHuPH20. — Initial 111 0.4 98.9 0.7 59 15 10 4.4 ffp13293 Skg 5° C. 1 week nd 0.4 98.7 0.9 Nd nd nd 5.2 ffp 13530 Skg 25° C.1 week nd 0.4 98.7 0.8 61 12 12 5.2 ffp  9390 F/T (5 cycles) nd 0.4 98.70.9 Nd nd nd 5.5 ffp 12532  5° C. 8 weeks 109 0.4 98.7 0.9 60 15 11 5.1ffp 13508 21 weeks nd 0.5 98.8 0.8 Nd nd nd 5.3 ffp nd 36 weeks nd 0.498.8 0.8 Nd nd nd 4.5 ffp nd 25° C. 8 weeks 109 0.5 98.2 1.3 53 8 19 4.6ffp nd 21 weeks nd 0.7 97.6 1.8 Nd nd nd 5.2 effp nd 36 weeks nd 0.897.1 2.1 Nd nd nd 5.3 ffp nd 30° C. 8 weeks 109 0.6 97.7 1.7 45 6 26 4.8ffp 13394 21 weeks 107 0.9 96.2 2.9 Nd nd nd 5.3 ffp nd Formulation C isa liquid formulation with the composition 120 mg/ml Trastuzumab, 20 mML-histidine/HCL pH 5.5, 130 mM sodium chloride, 10 mM methionine, 0.04%polysorbate 20, 12′000 U/ml rHuPH20. — Initial 129 0.5 98.8 0.7 59 15 1024.2 ffp 12355 Skg 5° C. 1 week nd 0.5 98.6 0.9 Nd nd nd 26.0 ffp 13123Skg 25° C. 1 week nd 0.5 98.5 0.9 60 12 11 25.3 ffp 12209 F/T (5 cycles)nd 0.5 98.6 0.9 Nd nd nd 24.8 ffp 12576  5° C. 8 weeks 126 0.5 98.7 0.860 15 11 25.4 ffp 12463 21 weeks nd 0.6 98.4 1.0 62 13  9 23.6 ffp 1540936 weeks nd 0.6 98.6 0.8 59 12 12 26.3 ffp 13218 25° C. 8 weeks 125 0.798.0 1.3 54 9 18 25.0 ffp 13038 21 weeks nd 0.9 97.5 1.6 42 9 22 24.3ffp 14972 36 weeks nd 1.0 97.0 2.1 32 9 31 25.7 ffp 12028 30° C. 8 weeks125 0.8 97.6 1.6 46 8 25 23.0 ffp 12751 21 weeks 125 1.2 96.3 2.6 32 829 25.7 ffp nd Formulation D is a liquid formulation with thecomposition 120 mg/ml Trastuzumab, 20 mM L-histidine/HCl pH 5.5, 130 mMsodium chloride, 10 mM methionine, 0.06% polysorbate 80, 12′000 U/mlrHuPH20. — Initial 128 0.4 98.9 0.7 59 15 10 25.1 ffp 15178 Skg 5° C. 1week Nd 0.5 98.6 0.9 Nd nd nd 24.1 effp 12201 Skg 25° C. 1 week Nd 0.698.5 0.9 60 12 11 25.4 effp  8311 F/T (5 cycles) Nd 0.5 98.6 0.9 Nd ndnd 24.8 effp 11906  5° C. 8 weeks 125 0.5 98.7 0.8 60 15 11 25.0 ffp13238 21 weeks Nd 0.6 98.5 1.0 Nd nd nd 24.3 ffp nd 36 weeks Nd 0.6 98.60.8 Nd nd nd 26.1 ffp nd 25° C. 8 weeks 125 0.7 98.1 1.2 54 9 18 23.4ffp 12661 21 weeks Nd 0.9 97.3 1.8 Nd nd nd 24.5 ffp nd 36 weeks Nd 1.196.8 2.1 Nd nd nd 26.6 ffp nd 30° C. 8 weeks 124 0.9 97.5 1.7 45 8 2523.7 ffp 12182 21 weeks 125 1.3 96.1 2.6 Nd nd nd 24.2 effp ndFormulation E is a liquid formulation with the composition 120 mg/mlTrastuzumab, 20 mM L-histidine/HCl pH 5.5, 105 mM α,α-trehalosedihydrate, 65 mM sodium chloride, 10 mM methionine, 0.04% polysorbate20, 12′000 U/ml rHuPH20. — Initial 128 0.4 98.9 0.7 59 15 10 16.6 ffp13475 Skg 5° C. 1 week nd 0.5 98.6 0.9 Nd nd nd 17.2 ffp 12363 Skg 25°C. 1 week nd 0.5 98.5 1.0 60 12 11 17.2 effp 12793 F/T (5 cycles) nd 0.598.6 0.9 Nd nd nd 16.5 effp 12374  5° C. 8 weeks 125 0.5 98.7 0.8 60 1511 16.9 ffp 13086 21 weeks nd 0.5 98.6 0.9 61 13 11 16.4 ffp 14896 36weeks nd 0.5 98.7 0.8 59 12 12 16.5 ffp 13321 25° C. 8 weeks 125 0.798.1 1.2 53 9 18 15.4 ffp nd 21 weeks nd 0.8 97.5 1.7 41 8 29 19.2 ffp14730 36 weeks nd 0.9 97.0 2.1 32 9 32 17.4 ffp 12028 30° C. 8 weeks 1240.8 97.7 1.5 45 8 25 15.7 ffp 11745 21 weeks 123 1.1 96.3 2.6 32 8 2417.4 ffp nd Formulation F is a liquid formulation with the composition120 mg/ml Trastuzumab, 20 mM L-histidine/HCl pH 5.5, 105 mMα,α-trehalose dihydrate, 65 mM sodium chloride, 10 mM methionine, 0.06%polysorbate 80, 12′000 U/ml rHuPH20. — Initial 127 0.4 98.9 0.7 59 15 1016.7 effp 13069 Skg 5° C. 1 week nd 0.5 98.6 0.9 Nd nd nd 16.0 ffp 13188Skg 25° C. 1 week nd 0.5 98.6 0.9 60 12 11 15.8 ffp  9764 F/T (5 cycles)nd 0.5 98.7 0.8 Nd nd nd 18.5 ffp 11769  5° C. 8 weeks 125 0.5 98.7 0.860 15 11 16.5 ffp nd 21 weeks nd 0.6 98.6 0.9 Nd nd nd 16.6 ffp nd 36weeks nd 0.6 98.7 0.8 Nd nd nd 17.2 ffp nd 25° C. 8 weeks 125 0.7 98.11.2 53 9 18 17.4 ffp 13570 21 weeks nd 0.9 97.4 1.7 Nd nd nd 16.4 ffp nd36 weeks nd 1.0 96.9 2.1 Nd nd nd 18.3 ffp nd 30° C. 8 weeks 124 0.897.5 1.7 45 8 25 16.2 ffp 11860 21 weeks 123 1.8 95.6 2.9 Nd nd nd 16.1ffp nd Formulation G is a liquid formulation with the composition 120mg/ml Trastuzumab, 20 mM L-histidine/HCl pH 5.5, 210 mM α,α-trehalosedihydrate, 10 mM methionine, 0.04% polysorbate 20. — Initial 122 0.399.7 nd 71 11  7 4.2 ffp  5° C. 4 weeks nd 0.3 99.6 nd 60 11 11 nd nd 12weeks 118 0.4 99.6 <0.3 70 10  7 4.8 ffp 24 weeks 121 0.4 99.5 nd 68 9 9 4.1 ffp 36 weeks nd 0.5 99.5 0.1 67 8 10 nd nd 15° C. 4 weeks nd 0.499.6 nd 70 10  7 nd nd 12 weeks 121 0.5 99.4 <0.3 66 8 11 4.3 ffp 24weeks 121 0.5 99.3 <0.3 59 7 17 4.4 ffp 36 weeks nd 0.6 99.2 0.2 53 7 22nd nd 25° C. 4 weeks nd 0.5 99.5 <0.3 66 7 11 nd nd 12 weeks 121 0.697.2 2.1 53 6 22 4.2 ffp 24 weeks 122 0.7 98.0 1.4 39 6 32 4.2 ffp andrHuPH20 added prior injection from a bulk with the followingcomposition: 10 mg/ml rHuPH20 in 20 mM His/Histidine/HCl, pH 6.5, 130 mMNaCl, 0.05% (w/v) polysorbate 80 Protein Size Exclusion-HPLC IonExchange-HPLC HE Storage Storage concentration HMW Monomer LMW Main PeakPeak 1 Peak 4 Turbidity Visible activity condition Time (mg/ml) (%) (%)(%) (%) (%) (%) (FTU) particles (U/ml) Formulation H is a liquidformulation with the composition 110 mg/ml Trastuzumab, 20 mML-histidine/HCl pH 5.0, 210 mM α,α-trehalose dihydrate, 5 mM methionine,0.02% polysorbate 20, 2′000 U/ml rHuPH20. — Initial 111 0.6 98.7 0.8 6912 6 3.9 ffp 2081 Skg 5° C. 1 week nd 0.6 98.7 0.7 68 12 6 3.9 ffp 2406Skg 25° C. 1 week nd 0.7 98.6 0.7 68 10 7 3.8 ffp nd F/T (5 cycles) nd0.7 98.7 0.7 68 12 6 3.8 ffp 2167  5° C. 8 weeks nd 0.6 98.8 0.6 69 10 84.0 ffp 2235 12 weeks 110 0.6 98.7 0.7 66 10 9 3.9 ffp 1970 36 weeks ndNd nd nd Nd nd nd nd nd nd 25° C. 8 weeks nd 0.7 98.3 1.0 57 5 15 4.1ffp 1891 12 weeks 111 0.7 97.8 1.5 50 4 20 3.8 ffp 2079 36 weeks nd ndnd nd Nd nd nd nd nd nd 40° C. 8 weeks nd 1.4 94.1 4.5 31 3 30 4.1 ffpnd 12 weeks 111 1.6 92.5 5.9 28 5 35 4.7 ffp nd Formulation I is aliquid formulation with the composition 110 mg/ml Trastuzumab, 20 mML-histidine/HCl pH 6.0, 210 mM α,α-trehalose dihydrate, 5 mM methionine,0.02% polysorbate 20, 2′000 U/ml rHuPH20. — Initial 111 0.8 98.5 0.7 6912 7 4.7 effp 1948 Skg 5° C. 1 week Nd 0.8 98.6 0.7 68 12 7 5.1 ffp 2672Skg 25° C. 1 week Nd 0.8 98.6 0.7 67 11 8 4.7 ffp 1724 F/T (5 cycles) Nd0.8 98.6 0.7 68 12 7 4.5 ffp 2507  5° C. 8 weeks Nd 0.8 98.6 0.6 68 11 84.5 ffp 1911 12 weeks 112 0.8 98.5 0.7 65 11 10 4.7 ffp 2034 36 weeks Ndnd nd nd nd nd nd nd nd nd 25° C. 8 weeks Nd 0.9 98.1 1.0 53 12 18 4.8effp 1910 12 weeks 111 1.0 97.8 1.2 45 12 21 4.9 ffp 2157 36 weeks Nd ndnd nd nd nd nd nd nd nd 40° C. 8 weeks Nd 1.6 95.3 3.1 26 9 26 5.0 ffpnd 12 weeks 112 1.9 93.9 4.3 20 9 22 5.7 ffp nd Formulation J is aliquid formulation with the composition 110 mg/ml Trastuzumab, 20 mML-histidine/HCl pH 5.0, 210 mM α,α-trehalose dihydrate, 5 mM methionine,0.06% polysorbate 20, 2′000 U/ml rHuPH20. — Initial 111 0.6 98.7 0.7 6912 6 3.7 ffp 2198 Skg 5° C. 1 week nd 0.6 98.6 0.7 68 12 6 4.3 effp 2528Skg 25° C. 1 week nd 0.6 98.6 0.8 68 10 7 4.0 effp 1993 F/T (5 cycles)nd 0.7 98.7 0.7 68 12 6 3.7 ffp 2256  5° C. 8 weeks nd 0.6 98.8 0.6 6810 8 4.1 ffp 2263 12 weeks 111 0.6 98.6 0.8 66 10 9 3.9 ffp 2337 36weeks nd nd nd nd Nd nd nd nd nd nd 25° C. 8 weeks nd 0.7 98.3 1.0 56 515 4.0 ffp 1931 12 weeks 111 0.7 97.9 1.4 50 4 20 4.0 ffp 2291 36 weeksnd nd nd nd Nd nd nd nd nd nd 40° C. 8 weeks nd 1.3 94.5 4.2 30 3 35 4.2ffp nd 12 weeks 111 1.6 92.4 6.0 27 5 35 4.2 ffp nd Formulation K is aliquid formulation with the composition 110 mg/ml Trastuzumab, 20 mML-histidine/HCl pH 6.0, 210 mM α,α-trehalose dihydrate, 5 mM methionine,0.06% polysorbate 20, 2′000 U/ml rHuPH20. — Initial 111 0.8 98.6 0.7 6912 7 4.7 ffp 2258 Skg 5° C. 1 week nd 0.8 98.6 0.7 68 12 7 5.0 ffp 2680Skg 25° C. 1 week nd 0.8 98.5 0.7 67 11 8 4.6 effp 2049 F/T (5 cycles)nd 0.8 98.6 0.7 69 12 7 4.8 ffp 2316  5° C. 8 weeks nd 0.8 98.5 0.7 6812 8 4.5 ffp 2132 12 weeks 112 0.8 98.5 0.7 65 12 9 5.2 ffp 2260 36weeks nd nd nd nd Nd nd nd nd nd nd 25° C. 8 weeks nd 0.9 98.3 0.8 53 1218 4.5 ffp 1863 12 weeks 112 1.0 97.9 1.2 46 12 21 4.7 ffp 1917 36 weeksnd nd nd nd Nd nd nd nd nd nd 40° C. 8 weeks nd 1.5 95.4 3.1 26 10 265.2 ffp nd 12 weeks 112 1.9 93.9 4.3 28 8 22 6.2 ffp nd Formulation L isa liquid formulation with the composition 110 mg/ml Trastuzumab, 20 mML-histidine/HCl pH 5.0, 210 mM α,α-trehalose dihydrate, 15 mMmethionine, 0.02% polysorbate 20, 2′000 U/ml rHuPH20. — Initial 111 0.698.7 0.7 69 12 6 3.5 effp 2301 Skg 5° C. 1 week nd 0.6 98.7 0.7 69 12 64.1 ffp 2574 Skg 25° C. 1 week nd 0.6 98.7 0.7 68 10 7 4.1 ffp nd F/T (5cycles) nd 0.7 98.5 0.8 68 12 7 3.6 ffp 2435  5° C. 8 weeks nd 0.6 98.80.6 68 10 7 3.8 ffp 2263 12 weeks 111 0.6 98.7 0.7 67 10 8 3.8 ffp 185736 weeks nd nd nd nd Nd nd nd nd nd nd 25° C. 8 weeks nd 0.7 98.4 1.0 565 15 3.8 ffp 1919 12 weeks 111 0.7 97.9 1.4 50 4 19 3.8 ffp 2106 36weeks nd nd nd nd Nd nd nd nd nd nd 40° C. 8 weeks nd 1.3 94.2 4.5 31 434 3.9 ffp nd 12 weeks 111 1.5 92.6 6.0 28 4 35 4.2 ffp nd Formulation Mis a liquid formulation with the composition 110 mg/ml Trastuzumab, 20mM L-histidine/HCl pH 6.0, 210 mM α,α-trehalose dihydrate, 15 mMmethionine, 0.02% polysorbate 20, 2′000 U/ml rHuPH20. — Initial 110 0.798.6 0.7 69 12 7 4.4 effp 2203 Skg 5° C. 1 week nd 0.7 98.6 0.6 69 12 75.2 ffp 2169 Skg 25° C. 1 week nd 0.7 98.6 0.7 67 11 8 4.9 ffp 1661 F/T(5 cycles) nd 0.7 98.6 0.6 68 12 7 4.6 ffp 2183  5° C. 8 weeks nd 0.798.6 0.7 68 12 8 4.5 ffp 2188 12 weeks 111 0.8 98.6 0.7 66 12 9 4.9 effp2028 36 weeks nd nd nd nd Nd nd nd nd nd nd 25° C. 8 weeks nd 0.9 98.11.1 53 12 18 4.5 ffp 1900 12 weeks 111 0.9 98.0 1.2 46 12 21 4.7 ffp1936 36 weeks nd nd nd nd Nd nd nd nd nd nd 40° C. 8 weeks nd 1.5 95.03.5 26 10 26 5.4 ffp nd 12 weeks 111 1.7 94.1 4.2 27 8 22 5.5 ffp ndFormulation N is a liquid formulation with the composition 110 mg/mlTrastuzumab, 20 mM L-histidine/HCl pH 5.0, 210 mM α,α-trehalosedihydrate, 15 mM methionine, 0.06% polysorbate 20, 2′000 U/ml rHuPH20. —Initial 111 0.6 98.7 0.7 69 12 7 3.8 effp 2410 Skg 5° C. 1 week Nd 0.698.6 0.7 69 12 6 4.0 effp 2559 Skg 25° C. 1 week Nd 0.6 98.6 0.8 68 10 73.7 ffp 2086 F/T (5 cycles) Nd 0.7 98.7 0.7 68 12 6 4.0 ffp 2457  5° C.8 weeks Nd 0.6 98.8 0.7 69 10 7 3.9 ffp 2102 12 weeks 111 0.6 98.8 0.767 10 8 3.6 effp 2037 36 weeks Nd nd nd nd Nd nd nd nd nd nd 25° C. 8weeks Nd 0.7 98.2 1.1 57 5 15 3.8 ffp 2215 12 weeks 111 0.7 97.9 1.4 504 19 3.7 ffp 2050 36 weeks Nd nd nd nd Nd nd nd nd nd nd 40° C. 8 weeksNd 1.3 94.7 4.1 30 4 34 4.0 ffp nd 12 weeks 111 1.5 92.5 6.0 28 4 35 4.5ffp nd Formulation O is a liquid formulation with the composition 110mg/ml Trastuzumab, 20 mM L-histidine/HCl pH 6.0, 210 mM α,α-trehalosedihydrate, 15 mM methionine, 0.06% polysorbate 20, 2′000 U/ml rHuPH20. —Initial 111 0.8 98.6 0.7 69 12 7 4.5 ffp 2153 Skg 5° C. 1 week nd 0.798.6 0.7 69 12 7 4.7 ffp 1846 Skg 25° C. 1 week nd 0.7 98.5 0.7 67 11 84.9 effp 2192 F/T (5 cycles) nd 0.7 98.6 0.7 69 12 7 4.3 effp 2323  5°C. 8 weeks nd 0.7 98.7 0.6 68 12 8 4.8 ffp 2049 12 weeks 112 0.8 98.50.7 66 12 9 4.6 ffp 1903 36 weeks nd nd nd nd Nd nd nd nd nd nd 25° C. 8weeks nd 0.9 98.2 0.9 53 12 18 4.8 ffp 2002 12 weeks 112 0.9 97.9 1.2 4613 21 4.8 ffp 2216 36 weeks nd nd nd nd Nd nd nd nd nd nd 40° C. 8 weeksnd 1.5 95.6 2.9 26 10 25 5.2 ffp nd 12 weeks 112 1.9 94.0 4.3 27 8 226.1 ffp nd Formulation P is a liquid formulation with the composition130 mg/ml Trastuzumab, 20 mM L-histidine/HCl pH 5.0, 210 mMα,α-trehalose dihydrate, 5 mM methionine, 0.02% polysorbate 20, 2′000U/ml rHuPH20. — Initial 128 0.7 98.7 0.7 69 12 6 3.8 ffp 2035 Skg 5° C.1 week Nd 0.7 98.7 0.7 69 12 6 3.4 ffp 2728 Skg 25° C. 1 week Nd 0.798.6 0.7 68 10 7 3.9 ffp nd F/T (5 cycles) Nd 0.7 98.7 0.7 69 12 6 3.7ffp 2559  5° C. 8 weeks Nd 0.6 98.8 0.6 69 10 7 3.6 ffp 2217 12 weeks129 0.6 98.7 0.7 67 11 9 3.8 ffp 1878 36 weeks Nd nd nd nd Nd nd nd ndnd nd 25° C. 8 weeks Nd 0.8 98.2 1.0 57 5 15 3.5 ffp 2091 12 weeks 1280.8 97.8 1.4 51 4 19 4.3 ffp 1887 36 weeks Nd nd nd nd Nd nd nd nd nd nd40° C. 8 weeks Nd 1.6 94.2 4.2 31 4 35 3.9 ffp nd 12 weeks 129 1.9 92.15.9 28 5 35 4.5 ffp nd Formulation Q is a liquid formulation with thecomposition 130 mg/ml Trastuzumab, 20 mM L-histidine/HCl pH 6.0, 210 mMα,α-trehalose dihydrate, 5 mM methionine, 0.02% polysorbate 20, 2′000U/ml rHuPH20. — Initial 131 0.8 98.5 0.7 69 12 7 4.4 effp 2309 Skg 5° C.1 week Nd 0.8 98.5 0.7 69 12 7 4.4 ffp 2522 Skg 25° C. 1 week Nd 0.998.4 0.7 67 11 8 4.8 ffp 1787 F/T (5 cycles) Nd 0.8 98.5 0.6 69 12 6 4.8ffp 2312  5° C. 8 weeks Nd 0.9 98.6 0.6 68 12 8 5.1 ffp 2131 12 weeks132 0.9 98.4 0.7 66 11 9 4.9 effp 1931 36 weeks Nd nd nd nd Nd nd nd ndnd nd 25° C. 8 weeks Nd 1.1 97.8 1.1 54 12 18 5.1 ffp 1888 12 weeks 1321.1 97.8 1.1 46 12 21 4.7 ffp 1912 36 weeks Nd nd nd nd Nd nd nd nd ndnd 40° C. 8 weeks Nd 1.8 95.2 3.0 27 10 27 5.6 effp nd 12 weeks 132 2.293.4 4.4 28 8 22 6.1 ffp nd Formulation R is a liquid formulation withthe composition 130 mg/ml Trastuzumab, 20 mM L-histidine/HCl pH 5.0, 210mM α,α-trehalose dihydrate, 5 mM methionine, 0.06% polysorbate 20, 2′000U/ml rHuPH20. — Initial 131 0.7 98.6 0.7 69 12 6 3.7 effp 2096 Skg 5° C.1 week Nd 0.7 98.7 0.6 69 12 6 3.7 effp 1856 Skg 25° C. 1 week Nd 0.798.5 0.8 68 10 7 4.3 effp 1958 F/T (5 cycles) Nd 0.7 98.7 0.6 69 12 63.8 effp 2371  5° C. 8 weeks Nd 0.7 98.7 0.6 69 10 7 3.5 effp 2075 12weeks 131 0.6 98.6 0.7 67 10 8 3.6 effp 2350 36 weeks Nd nd nd nd Nd ndnd nd nd nd 25° C. 8 weeks Nd 0.9 98.2 1.0 58 5 15 3.5 effp 1989 12weeks 131 0.9 97.7 1.4 51 4 20 3.9 effp 1999 36 weeks Nd nd nd nd Nd ndnd nd nd nd 40° C. 8 weeks Nd 1.6 94.3 4.1 32 4 35 4.2 ffp nd 12 weeks132 2.0 92.0 6.0 29 5 35 4.6 effp nd Formulation S is a liquidformulation with the composition 130 mg/ml Trastuzumab, 20 mML-histidine/HCl pH 6.0, 210 mM α,α-trehalose dihydrate, 5 mM methionine,0.06% polysorbate 20, 2′000 U/ml rHuPH20. — Initial 131 0.8 98.5 0.7 6912 7 4.6 effp 2406 Skg 5° C. 1 week Nd 0.8 98.5 0.7 69 12 7 4.8 wafp2808 Skg 25° C. 1 week Nd 0.9 98.4 0.7 67 11 8 4.9 ffp 2141 F/T (5cycles) Nd 0.8 98.5 0.6 69 12 6 4.7 ffp 2487  5° C. 8 weeks Nd 0.8 98.50.7 69 12 8 4.8 effp 2076 12 weeks 132 0.9 98.4 0.7 66 12 9 4.8 effp1897 36 weeks Nd nd nd nd Nd nd nd nd nd nd 25° C. 8 weeks Nd 1.1 97.81.1 55 12 18 5.0 ffp 1956 12 weeks 131 1.1 97.7 1.2 47 12 21 4.9 ffp2094 36 weeks Nd nd nd nd Nd nd nd nd nd nd 40° C. 8 weeks Nd 1.9 95.23.0 27 9 27 5.7 ffp nd 12 weeks 133 2.2 93.5 4.3 21 9 22 6.1 ffp ndFormulation T is a liquid formulation with the composition 130 mg/mlTrastuzumab, 20 mM L-histidine/HCl pH 5.0, 210 mM α,α-trehalosedihydrate, 15 mM methionine, 0.02% polysorbate 20, 2′000 U/ml rHuPH20. —Initial 131 0.7 98.7 0.7 69 12 6 3.7 effp 2311 Skg 5° C. 1 week nd 0.798.7 0.7 69 12 6 3.7 ffp 2397 Skg 25° C. 1 week nd 0.7 98.5 0.8 68 10 74.0 effp nd F/T (5 cycles) nd 0.7 98.7 0.6 68 12 6 3.7 ffp 2277  5° C. 8weeks nd 0.6 98.8 0.5 67 10 7 3.5 ffp 2167 12 weeks 131 0.6 98.6 0.7 6910 7 3.7 ffp 2070 36 weeks nd nd nd nd Nd nd nd nd nd nd 25° C. 8 weeksnd 0.8 98.2 1.0 56 5 15 3.6 ffp 2122 12 weeks 131 0.8 97.8 1.4 52 4 193.7 ffp 2138 36 weeks nd nd nd nd Nd nd nd nd nd nd 40° C. 8 weeks nd1.5 94.3 4.2 31 4 34 4.0 ffp nd 12 weeks 132 1.8 92.2 6.0 29 4 35 4.1ffp nd Formulation U is a liquid formulation with the composition 130mg/ml Trastuzumab, 20 mM L-histidine/HCl pH 6.0, 210 mM α,α-trehalosedihydrate, 15 mM methionine, 0.02% polysorbate 20, 2′000 U/ml rHuPH20. —Initial 131 0.8 98.5 0.7 69 12 7 4.5 effp 2768 Skg 5° C. 1 week nd 0.898.6 0.7 68 12 7 4.7 effp 2884 Skg 25° C. 1 week nd 0.8 98.5 0.7 68 11 86.0 effp 2044 F/T (5 cycles) nd 0.8 98.6 0.6 68 12 7 4.6 ffp 2617  5° C.8 weeks nd 0.8 98.5 0.7 67 12 8 4.3 ffp 2571 12 weeks 132 0.8 98.5 0.767 12 8 4.5 ffp 2164 36 weeks nd nd nd nd Nd nd nd nd nd nd 25° C. 8weeks nd 1.0 98.0 1.0 52 12 18 4.9 ffp 2116 12 weeks 131 1.0 97.7 1.3 4713 21 5.0 effp 1990 36 weeks nd nd nd nd Nd nd nd nd nd nd 40° C. 8weeks nd 1.7 95.4 2.9 24 10 24 5.4 ffp nd 12 weeks 132 2.0 93.7 4.3 19 920 5.7 ffp nd Formulation V is a liquid formulation with the composition130 mg/ml Trastuzumab, 20 mM L-histidine/HCl pH 5.0, 210 mMα,α-trehalose dihydrate, 15 mM methionine, 0.06% polysorbate 20, 2′000U/ml rHuPH20. — Initial 131 0.7 98.7 0.7 68 12 6 3.7 effp 2323 Skg 5° C.1 week nd 0.7 98.6 0.7 69 12 6 3.7 ffp 2646 Skg 25° C. 1 week nd 0.798.5 0.8 68 10 7 3.8 ffp 2056 F/T (5 cycles) nd 0.7 98.7 0.7 68 12 6 3.5effp 2498  5° C. 8 weeks nd 0.6 98.9 0.5 67 10 7 3.5 effp 2179 12 weeks132 0.6 98.6 0.8 69 10 7 3.7 ffp 2119 36 weeks nd nd nd nd Nd nd nd ndnd nd 25° C. 8 weeks nd 0.8 98.1 1.1 56 5 15 3.6 ffp 2072 12 weeks 1320.8 97.8 1.4 52 4 19 3.9 effp 2348 36 weeks nd nd nd nd Nd nd nd nd ndnd 40° C. 8 weeks nd 1.5 94.2 4.3 31 4 34 3.9 ffp nd 12 weeks 132 1.892.3 5.9 30 5 34 4.3 ffp nd Formulation W is a liquid formulation withthe composition 130 mg/ml Trastuzumab, 20 mM L-histidine/HCl pH 6.0, 210mM α,α-trehalose dihydrate, 15 mM methionine, 0.06% polysorbate 20,2′000 U/ml rHuPH20. — Initial 131 0.8 98.5 0.7 69 12 7 4.7 ffp 2018 Skg5° C. 1 week nd 0.8 98.5 0.7 69 12 7 4.6 ffp 1790 Skg 25° C. 1 week nd0.8 98.5 0.7 67 11 8 4.9 ffp 1918 F/T (5 cycles) nd 0.8 98.6 0.7 69 12 74.7 ffp 2379  5° C. 8 weeks nd 0.8 98.7 0.5 67 11 8 4.4 ffp 2028 12weeks 131 0.8 98.4 0.8 67 12 8 4.7 effp 1964 36 weeks nd nd nd nd Nd ndNd nd nd nd 25° C. 8 weeks nd 1.0 98.0 1.0 53 12 18 4.3 ffp 2198 12weeks 131 1.0 97.8 1.2 47 13 20 5.0 ffp 1894 36 weeks nd nd nd nd Nd ndNd nd nd nd 40° C. 8 weeks nd 1.7 95.4 3.0 24 9 25 5.0 ffp nd 12 weeks132 2.0 93.7 4.4 27 9 20 5.8 effp nd Formulation X is a liquidformulation with the composition 120 mg/ml Trastuzumab, 20 mML-histidine/HCl pH 5.5, 210 mM α,α-trehalose dihydrate, 10 mMmethionine, 0.04% polysorbate 20, 2′000 U/ml rHuPH20. — Initial 121 0.898.5 0.7 69 12 7 4.2 effp 2277 Skg 5° C. 1 week nd 0.8 98.6 0.7 69 12 64.2 ffp 1855 Skg 25° C. 1 week nd 0.8 98.5 0.7 68 11 7 4.8 ffp 2070 F/T(5 cycles) nd 0.8 98.6 0.7 69 12 6 4.5 ffp 2477  5° C. 8 weeks nd 0.898.6 0.6 68 10 8 4.3 ffp 2447 12 weeks 122 0.7 98.6 0.7 67 10 9 4.3 ffp2189 36 weeks nd nd nd nd Nd nd Nd nd nd nd 25° C. 8 weeks nd 0.9 98.21.0 57 7 18 4.5 ffp 2030 12 weeks 122 0.9 97.8 1.3 50 7 23 4.5 ffp 203036 weeks nd nd nd nd Nd nd Nd nd nd nd 40° C. 8 weeks nd 1.4 95.3 3.3 346 33 4.8 ffp nd 12 weeks 122 1.7 93.6 4.6 32 7 31 5.1 ffp nd

Example 2: Preparation of a Lyophilized Formulation

A solution of approx. 60 mg/ml Trastuzumab was prepared as describedabove for liquid formulations. All excipients have been added at half ofthe concentration of the above mentioned liquid formulation. Theformulation was sterile filtered through 0.22 μm filters and asepticallydistributed in equal amounts into sterile 20 ml glass vials. The vialswere partly closed with ETFE (Copolymer of ethylene andtetrafluoroethylene)-coated rubber stoppers suitable for the use inlyophilization processes and lyophilized using the freeze-drying cyclereported in Table 2.

TABLE 2 Freeze-drying Cycle Shelf Ramp Hold Vacuum temperature Rate timeSet point Step (° C.) (° C./min) (min) (μbar) Pre-cooling    5° C. 0.060 — Freezing −40° C. 1.0 120 — Primary Drying −25° C. 0.5 4560 80Secondary Drying +25° C. 0.2 300 80

The product was first cooled from room temperature to approx 5° C.(pre-cooling), followed by a freezing step at −40° C. with a platecooling rate of approx. 1° C./min, followed by a holding step at −40° C.for about 2 hours. The first drying step was performed at a platetemperature of approx. −25° C. and a chamber pressure of approx. 80 μbarfor about 76 hours. Subsequently, the second drying step started with atemperature ramp of 0.2° C./min from −25° C. to 25° C., followed by aholding step at 25° C. for at least 5 hours at a chamber pressure ofapprox. 80 μbar.

Lyophilization was carried out in a Usifroid SMH-90 LN2 freeze-dryer(Usifroid, Maurepas, France) or a LyoStar II Freeze-dryer (FTS Systems,Stone Ridge, N.Y., USA). The freeze-dried samples were stored atdifferent climate conditions (5° C., 25° C. and 30° C.) for differentintervals of time. The lyophilized vials were reconstituted to a finalvolume of 2.65 ml with water for injection (WFI) yielding an isotonicformulation with an antibody concentration of approx. 120 mg/ml. Thereconstitution time of the freeze-dried cakes was around 10 min.Analysis of the reconstituted samples was performed after a 24 hourincubation period of the reconstituted liquid sample at ambienttemperature.

The samples were again analyzed by the following analytical methodsdescribed above:

1) UV spectrophotometry;

2) Size Exclusion Chromatography (SEC);

3) Ion exchange chromatography (IEC);4) turbidity of the solution; and5) for visible particles.

The results of the stability testing for the Formulations is provided inthe Table 3 below wherein the following abbreviations are used:

ffp:=free from particles;effp:=essentially free from particles;nd:=not determined

TABLE 3 Composition and stability data of a lyophilized anti-HER2 drugproduct formulation according to this invention Formulation Y is alyophilized formulation with the composition 120 mg/ml Trastuzumab, 20mM L-histidine/HC1 pH 5.5, 210 mM α,α-trehalose dihydrate, 10 mMmethionine, 0.04% polysorbate 20, 12′000 U/ml rHuPH20 afterreconstitution. Protein Size Exclusion-HPLC Ion Exchange-HPLC HE StorageStorage concentra- HMW Monomer LMW Main Peak Peak 1 Peak 4 TurbidityVisible activity condition Time tion (mg/ml) (%) (%) (%) (%) (%) (%)(FTU) particles (U/ml) — Initial 129 0.5 98.8 0.8 59 15 10 7.1 ffp 12451 5° C. 8 weeks 124 0.6 98.6 0.9 59 16 10 5.7 effp 13380 21 weeks nd 0.898.6 0.7 62 15 8 5.9 effp 14927 36 weeks nd 0.8 98.5 0.7 59 16 10 6.0ffp 13744 25° C. 8 weeks 120 1.3 97.8 0.8 59 16 11 6.1 effp 13162 21weeks nd 2.1 96.7 1.2 61 15 8 5.7 effp 15396 36 weeks nd 2.7 96.6 0.7 5815 11 6.0 ffp 13673 30° C. 8 weeks 121 1.9 97.5 0.9 58 16 11 5.7 effp13425 21 weeks 126 3.2 96.0 0.8 60 14 8 6.0 effp nd 36 weeks nd 3.9 95.40.7 57 14 11 5.9 ffp 15034

The properties of the above formulations provided are summarized in thefollowing Table 4:

PS Tr His tre NaC1 Meth 20 80 rHu F mg/ml m pH mM mM mM % % U/ml A 12020 5.5 210 10 0.04 12′000 B 120 20 5.5 210 10 0.06 12′000 C 120 20 5.5130 10 0.04 12′000 D 120 20 5.5 130 10 0.06 12′000 E 120 20 5.5 105 6510 0.04 12′000 F 120 20 5.5 105 65 10 0.06 12′000 G 120 20 5.5 210 100.04 H 110 20 5.0 210 5 0.02 2′000 I 110 20 6.0 210 5 0.02 2′000 J 11020 5.0 210 5 0.06 2′000 K 110 20 6.0 210 5 0.06 2′000 L 110 20 5.0 21015 0.02 2′000 M 110 20 6.0 210 15 0.02 2′000 N 110 20 5.0 210 15 0.062′000 O 110 20 6.0 210 15 0.06 2′000 P 130 20 5.0 210 5 0.02 2′000 Q 13020 6.0 210 5 0.02 2′000 R 130 20 5.0 210 5 0.06 2′000 S 130 20 6.0 210 50.06 2′000 T 130 20 5.0 210 15 0.02 2′000 U 130 20 6.0 210 15 0.02 2′000V 130 20 5.0 210 15 0.06 2′000 W 130 20 6.0 210 15 0.06 2′000 X 120 205.5 210 10 0.04 2′000 Y 120 20 5.5 210 10 0.04 12′000 *) F = FormulationTr = Trastuzumab His = L-histidine/HC1 tre = α,α-trehalose dihydrateNaCl = Sodium Chloride met = methionine PS = polysorbate in % (w/v) rHu= rHuPH20 *) = after reconstitution

What is claimed is:
 1. A highly concentrated, stable pharmaceuticalformulation of a pharmaceutically active anti-HER2 antibody comprising:a. about 50 to 350 mg/ml anti-HER2 antibody; b. about 1 to 100 mM of abuffering agent providing a pH of 5.5±2.0; c. about 1 to 500 mM of astabilizer or a mixture of two or more stabilizers; d. about 0.01 to0.08% of a nonionic surfactant; and e. an effective amount of at leastone hyaluronidase enzyme.
 2. The formulation according to claim 1,wherein the anti-HER2 antibody concentration is about 100 to 150 mg/ml.3. The formulation according to claim 2, wherein the anti-HER2 antibodyconcentration is 120±18 mg/ml.
 4. The formulation according to claim 1,comprising more than 150 to about 16'000 U/ml of a hyaluronidase enzyme.5. The formulation according to claim 4, comprising about 2'000 U/ml toabout 12'000 U/ml of a hyaluronidase enzyme.
 6. The formulationaccording to claim 1, wherein the buffering agent is at a concentrationof 1 to 50 mM.
 7. The formulation according to claim 6, wherein saidbuffering agent is a histidine buffer.
 8. The formulation according toclaim 1, wherein the formulation includes α,α-trehalose dihydrate orsucrose as a stabilizer.
 9. The formulation according to claim 1,wherein the stabilizer is at a concentration of 15 to 250 mM.
 10. Theformulation according to claim 8, wherein methionine is used as a secondstabilizer in a concentration of 5 to 25 mM.
 11. The formulationaccording to claim 1, wherein the nonionic surfactant is a polysorbateselected from the group consisting of polysorbate 20, polysorbate 80,and polyethylene-polypropylene copolymer.
 12. The formulation accordingto claim 11, wherein the concentration of the polysorbate is 0.02%(w/v), 0.04% (w/v) or 0.06% (w/v), respectively.
 13. The formulationaccording to claim 1, wherein the anti-HER2 antibody is selected fromthe group of Trastuzumab, Pertuzumab and T-DM1 or a combination of suchantibodies.
 14. The formulation according to claim 1 which is stableupon freezing and thawing.
 15. The formulation according to claim 1,wherein the hyaluronidase enzyme is rHuPH20.
 16. The formulationaccording to claim 1, wherein the buffering agent provides a pH of5.5±0.5.
 17. The formulation according to claim 1 for subcutaneous orintramuscular administration.
 18. The formulation according to claim 1in liquid form.
 19. The formulation according to claim 1 in lyophilizedform.
 20. A highly concentrated, stable pharmaceutical formulation of apharmaceutically active anti-HER2 antibody comprising: a. about 50 to350 mg/ml anti-HER2 antibody; b. about 1 to 100 mM of a buffering agentproviding a pH of 5.5±2.0; c. about 1 to 500 mM of a stabilizer or amixture of two or more stabilizers; and d. about 0.01 to 0.08% of anonionic surfactant.
 21. The formulation according to claim 1 for thetreatment of a disease or disorder amenable to treatment with ananti-HER2 antibody.
 22. An injection device comprising a highlyconcentrated, stable pharmaceutical anti-HER2 antibody formulationaccording to claim
 1. 23. The injection device according to claim 22wherein the formulation is co-administered concomitantly or sequentiallywith a chemotherapeutic agent.
 24. A method of treating a disease ordisorder which is amenable to treatment with an anti-HER2 antibody suchas cancer or a non-malignant disease in a subject comprisingadministering a formulation according to claim 1 to a subject in anamount effective to treat the said disease or disorder.
 25. The methodof claim 24 wherein said disease is cancer.
 26. A kit comprising one ormore vials containing the formulation according to claim 1 andinstructions for subcutaneous administration of the formulation to apatient.
 27. The kit according to claim 26 further comprising aninjection device for subcutaneous administration of the formulation to apatient.